Drought tolerant plants and related constructs and methods involving genes encoding ferrochelatases

ABSTRACT

Isolated polynucleotides and polypeptides and recombinant DNA constructs useful for conferring drought tolerance, compositions (such as plants or seeds) comprising these recombinant DNA constructs, and methods utilizing these recombinant DNA constructs. The recombinant DNA construct comprises a polynucleotide operably linked to a promoter that is functional in a plant, wherein said polynucleotide encodes a ferrochelatase.

This application is a divisional of U.S. patent application Ser. No.12/887,724, filed Sep. 22, 2010, now U.S. Pat. No. 8,158,859, which is adivisional of U.S. patent application Ser. No. 12/326,206, filed Dec. 2,2008, now U.S. Pat. No. 7,812,223, which claims the benefit of U.S.Provisional Application No. 60/991,859, filed Dec. 3, 2007, the entirecontent of each is herein incorporated by reference.

FIELD OF THE INVENTION

The field of invention relates to plant breeding and genetics and, inparticular, relates to recombinant DNA constructs useful in plants forconferring tolerance to drought.

BACKGROUND OF THE INVENTION

Abiotic stressors significantly limit crop production worldwide.Cumulatively, these factors are estimated to be responsible for anaverage 70% reduction in agricultural production (Bresson, 1999).

Drought stress, in particular, not only causes a reduction in theaverage yield for crops but also causes yield instability through highinterannual yield variation. Globally, about 35-40% of arable land fallsunder arid or semiarid classification. Even in non-arid regions wheresoils are nutrient-rich, drought stress occurs regularly for briefperiods or at moderate levels. Moreover, it has been predicted that inthe coming years rainfall patterns will shift and become more variabledue to increased global temperatures.

U.S. studies have shown that the ten most important kinds of cultivatedplants (corn, soybeans, wheat, tomatoes, etc.) produced only about 50%of the genetically possible yields on average per year; two thirds ofthe losses were due to the frequent combination of heat stress and watershortage (G. Schütte, S. Stirn, and V. Beusmann, TransgenePflanzen—Sicherheitsforschung, Risikoabschätzung andNachzulassungs-Monitoring. Birkhäuser Verlag A G, Basel-Boston-Berlin,2001).

Plants are sessile and have to adjust to the prevailing environmentalconditions of their surroundings. This has led to their development of agreat plasticity in gene regulation, morphogenesis, and metabolism.Adaptation and defense strategies involve the activation of genesencoding proteins important in the acclimation or defense towards thedifferent stressors. Some of the molecular responses to abiotic stressfactors such as drought are specific, but it has also been shown thatsimilar genes are activated by several stressors (Royal Society ofLondon, Transgenic Plants and World Agriculture, 2000, National AcademyPress, Washington, D.C.). It is believed that about 15 percent of aplant's genome is devoted to stress perception and adaptation (see e.g.,Cushman and Bohnert, 2000).

Earlier work on molecular aspects of abiotic stress responses wasaccomplished by differential and/or subtractive analysis (e.g., seeBray, 1993, Shinozaki and Yamaguchi-Shinozaki, 1997, Zhu et al., 1997,Thomashow, 1999). Other methods include selection of candidate genes(e.g., selection of genes from a particular known module and analyzingexpression of such a gene or its active product under stresses, or byfunctional complementation in a stressor system that is well defined,see Xiong and Zhu, 2001). Additionally, forward and reverse geneticstudies involving the identification and isolation of mutations inregulatory genes have also been used to provide evidence for observedchanges in gene expression under stress or exposure (Xiong and Zhu,2001).

Activation tagging can be utilized to identify genes with the ability toaffect a trait. This approach has been used in the model plant speciesArabidopsis thaliana (Weigel et al., Plant Physiol. 122:1003-1013(2000)). Insertions of transcriptional enhancer elements can dominantlyactivate and/or elevate the expression of nearby endogenous genes. Thismethod can be used to select genes involved in agronomically importantphenotypes, including stress tolerance.

SUMMARY OF THE INVENTION

The present invention includes:

In one embodiment, a plant comprising in its genome a recombinant DNAconstruct comprising a polynucleotide operably linked to at least oneregulatory element, wherein said polynucleotide encodes a polypeptidehaving an amino acid sequence of at least 50% sequence identity, basedon the Clustal V method of alignment, when compared to SEQ ID NO:2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, and wherein saidplant exhibits increased drought tolerance when compared to a controlplant not comprising said recombinant DNA construct.

In another embodiment, a method of increasing drought tolerance in aplant, comprising (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory sequence, wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 50% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct and exhibits increased drought tolerance whencompared to a control plant not comprising the recombinant DNAconstruct; and optionally, (c) obtaining a progeny plant derived fromthe transgenic plant, wherein said progeny plant comprises in its genomethe recombinant DNA construct and exhibits increased drought tolerancewhen compared to a control plant not comprising the recombinant DNAconstruct.

In another embodiment, a method of evaluating drought tolerance in aplant, comprising (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory sequence, wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 50% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct; and (c) evaluating the transgenic plant fordrought tolerance compared to a control plant not comprising therecombinant DNA construct; and optionally, (d) obtaining a progeny plantderived from the transgenic plant, wherein the progeny plant comprisesin its genome the recombinant DNA construct; and optionally, (e)evaluating the progeny plant for drought tolerance compared to a controlplant not comprising the recombinant DNA construct.

In another embodiment, a method of evaluating drought tolerance in aplant, comprising (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory sequence, wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 50% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct; (c) obtaining a progeny plant derived fromthe transgenic plant, wherein the progeny plant comprises in its genomethe recombinant DNA construct; and (d) evaluating the progeny plant fordrought tolerance compared to a control plant not comprising therecombinant DNA construct.

In another embodiment, the present invention includes any of the methodsof the present invention wherein the plant is a maize plant or a soybeanplant.

In another embodiment, the present invention includes an isolatedpolynucleotide comprising: (a) a nucleotide sequence encoding aferrochelatase, wherein the polypeptide has an amino acid sequence of atleast 90% or 95% sequence identity, based on the Clustal V method ofalignment, when compared to one of SEQ ID NO:4, 6, 8, 10, 12, 14, 16,18, 24, 26, 28 or 30, or (b) a full complement of the nucleotidesequence, wherein the full complement and the nucleotide sequenceconsist of the same number of nucleotides and are 100% complementary.The polypeptide may comprise the amino acid sequence of SEQ ID NO:4, 6,8, 10, 12, 14, 16, 18, 24, 26, 28 or 30. The nucleotide sequence maycomprise the nucleotide sequence of SEQ ID NO:3, 5, 7, 9, 11, 13, 15,17, 23, 25, 27 or 29.

In another embodiment, the present invention concerns a recombinant DNAconstruct comprising any of the isolated polynucleotides of the presentinvention operably linked to at least one regulatory sequence, and acell, a plant, and a seed comprising the recombinant DNA construct.

In another embodiment, the present invention includes a vectorcomprising any of the isolated polynucleotides of the present invention.

In another embodiment, the present invention concerns a cell, plant orseed comprising any of the recombinant DNA constructs of the presentinvention. The cell may be eukaryotic, e.g., a yeast, insect or plantcell, or prokaryotic, e.g., a bacterium.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1 shows a schematic of the pHSbarENDs2 activation tagging construct(SEQ ID NO:31) used to make the Arabidopsis populations.

FIG. 2 shows a map of the vector pDONR™/Zeo (SEQ ID NO:32). The attP1site is at nucleotides 570-801; the attP2 site is at nucleotides2754-2985 (complementary strand).

FIG. 3 shows a map of the vector pDONR™221 (SEQ ID NO:33). The attP1site is at nucleotides 570-801; the attP2 site is at nucleotides2754-2985 (complementary strand).

FIG. 4 shows a map of the vector pBC-yellow (SEQ ID NO:34), adestination vector for use in construction of expression vectors forArabidopsis. The attR1 site is at nucleotides 11276-11399 (complementarystrand); the attR2 site is at nucleotides 9695-9819 (complementarystrand).

FIG. 5 shows a map of PHP27840 (SEQ ID NO:35), a destination vector foruse in construction of expression vectors for soybean. The attR1 site isat nucleotides 7310-7434; the attR2 site is at nucleotides 8890-9014.

FIG. 6 shows a map of PHP23236 (SEQ ID NO:36), a destination vector foruse in construction of expression vectors for Gaspe Flint derived maizelines. The attR1 site is at nucleotides 2006-2130; the attR2 site is atnucleotides 2899-3023.

FIG. 7 shows a map of PHP10523 (SEQ ID NO:37), a plasmid DNA present inAgrobacterium strain LBA4404 (Komari et al., Plant J. 10:165-174 (1996);NCBI General Identifier No. 59797027).

FIG. 8 shows a map of PHP23235 (SEQ ID NO:38), a vector used toconstruct the destination vector PHP23236.

FIG. 9 shows a map of PHP28647 (SEQ ID NO:39), a destination vector foruse with maize inbred-derived lines. The attR1 site is at nucleotides2289-2413; the attR2 site is at nucleotides 3869-3993.

FIGS. 10A-10C show the multiple alignment of the amino acid sequences ofthe ferrochelatase-I proteins of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16,18, 48, 49 and 50. Residues that are identical to the residue of SEQ IDNO:2 at a given position are enclosed in a box. A consensus sequence ispresented where a residue is shown if identical in all sequences,otherwise, a period is shown.

FIG. 11 shows the percent sequence identity and the divergence valuesfor each pair of amino acids sequences of ferrochelatases displayed inFIGS. 10A-10C.

FIGS. 12A-12C show the multiple alignment of the amino acid sequences ofthe ferrochelatase-II proteins of SEQ ID NOs:20, 22, 24, 26, 28, 30, 51,52, 53 and 54. Residues that are identical to the residue of SEQ IDNO:20 at a given position are enclosed in a box. A consensus sequence ispresented where a residue is shown if identical in all sequences,otherwise, a period is shown.

FIG. 13 shows the percent sequence identity and the divergence valuesfor each pair of amino acids sequences of ferrochelatases displayed inFIGS. 12A-12C.

FIGS. 14A-14B show an evaluation of individual Gaspe Flint derived maizelines transformed with PHP31419.

FIG. 15 shows a summary evaluation of Gaspe Flint derived maize linestransformed with PHP31419.

FIGS. 16A-16B show an evaluation of individual Gaspe Flint derived maizelines transformed with PHP33089.

FIG. 17 shows a summary evaluation of Gaspe Flint derived maize linestransformed with PHP33089.

FIGS. 18A-18B show an evaluation of individual Gaspe Flint derived maizelines transformed with PHP30829.

FIG. 19 shows a summary evaluation of Gaspe Flint derived maize linestransformed with PHP30829.

FIGS. 20A-20B show an evaluation of individual Gaspe Flint derived maizelines transformed with PHP30745.

FIG. 21 shows a summary evaluation of Gaspe Flint derived maize linestransformed with PHP30745.

FIGS. 22A-22B show an evaluation of individual Gaspe Flint derived maizelines transformed with PHP30761.

FIG. 23 shows a summary evaluation of Gaspe Flint derived maize linestransformed with PHP30761.

SEQ ID NO:1 corresponds to NCBI GI No. 511080 which is the nucleotidesequence of a cDNA fragment encoding an Arabidopsis ferrochelatase-Iprotein (locus At5g26030).

SEQ ID NO:2 corresponds to NCBI GI No. 511081, which is the amino acidsequence of the Arabidopsis ferrochelatase-I protein encoded by SEQ IDNO:1.

TABLE 1 cDNAs Encoding Ferrochelatase-I (FeC-I) SEQ ID NO: SEQ ID NO:Plant Clone Designation (Nucleotide) (Amino Acid) Sugar Beetebs1c.pk002.n16 (FIS) 3 4 Brassica ebb1c.pk006.j11 (FIS) 5 6 Maizecfp3n.pk004.f12 (FIS) 7 8 Maize cfp5n.pk009.j16 (FIS) 9 10 Ricerl0n.pk117.h21 (FIS) 11 12 Soybean se3.pk0034.e10 (FIS) 13 14 Tulipetb1n.pk002.n16 (FIS) 15 16 Wheat wlp1c.pk002.p10 (FIS) 17 18

SEQ ID NO:19 corresponds to NCBI GI No. 30684569 which is the nucleotidesequence of a cDNA fragment encoding a first allele of an Arabidopsisferrochelatase-II protein (locus At2g30390).

SEQ ID NO:20 corresponds to NCBI GI No. 15227742, which is the aminoacid sequence of the Arabidopsis ferrochelatase-II protein encoded bySEQ ID NO:19.

SEQ ID NO:21 corresponds to NCBI GI No. 2623989, which is the nucleotidesequence of a cDNA fragment encoding a second allele of an Arabidopsisferrochelatase-II protein (locus At2g30390).

SEQ ID NO:22 corresponds to NCBI GI No. 2623990, which is the amino acidsequence of the Arabidopsis ferrochelatase-II protein encoded by SEQ IDNO:21.

TABLE 2 cDNAs Encoding Ferrochelatase-II (FeC-II) SEQ ID NO: SEQ ID NO:Plant Clone Designation (Nucleotide) (Amino Acid) Catmintecl1c.pk005.l15 (FIS) 23 24 Maize cfp6n.pk072.n9 (FIS) 25 26 WheatWlp1c.pk004.d13 (FIS) 27 28 Wheat wpa1c.pk014.g4 (FIS) 29 30

SEQ ID NO:31 is the nucleotide sequence of the pHSbarENDs2 activationtagging vector.

SEQ ID NO:32 is the nucleotide sequence of the GATEWAY® donor vectorpDONR™/Zeo.

SEQ ID NO:33 is the nucleotide sequence of the GATEWAY® donor vectorpDONR™221.

SEQ ID NO:34 is the nucleotide sequence of pBC-yellow, a destinationvector for use with Arabidopsis.

SEQ ID NO:35 is the nucleotide sequence of PHP27840, a destinationvector for use with soybean.

SEQ ID NO:36 is the nucleotide sequence of PHP23236, a destinationvector for use with Gaspe Flint derived maize lines.

SEQ ID NO:37 is the nucleotide sequence of PHP10523 (Komari et al.,Plant J. 10:165-174 (1996); NCBI General Identifier No. 59797027).

SEQ ID NO:38 is the nucleotide sequence of PHP23235, a destinationvector for use with Gaspe Flint derived lines.

SEQ ID NO:39 is the nucleotide sequence of PHP28647, a destinationvector for use with maize inbred-derived lines.

SEQ ID NO:40 is the nucleotide sequence of the attB1 site.

SEQ ID NO:41 is the nucleotide sequence of the attB2 site.

SEQ ID NO:42 is the nucleotide sequence of the At5g26030-5′attB forwardprimer, containing the attB1 sequence, used to amplify the At5g26030protein-coding region.

SEQ ID NO:43 is the nucleotide sequence of the At5g26030-3′attB reverseprimer, containing the attB2 sequence, used to amplify the At5g26030protein-coding region.

SEQ ID NO:44 is the nucleotide sequence of the At2g30390-5′attB forwardprimer, containing the attB1 sequence, used to amplify the At2g30390protein-coding region.

SEQ ID NO:45 is the nucleotide sequence of the At2g30390-3′attB reverseprimer, containing the attB2 sequence, used to amplify the At2g30390protein-coding region.

SEQ ID NO:46 is the nucleotide sequence of the VC062 primer, containingthe T3 promoter and attB1 site, useful to amplify cDNA inserts clonedinto a Bluescript® II SK(+) vector (Stratagene).

SEQ ID NO:47 is the nucleotide sequence of the VC063 primer, containingthe T7 promoter and attB2 site, useful to amplify cDNA inserts clonedinto a Bluescript® II SK(+) vector (Stratagene).

SEQ ID NO:48 is the amino acid sequence of a Hordeum vulgareferrochelatase-I protein (NCBI GI NO. 2460251).

SEQ ID NO:49 is the amino acid sequence of a rice ferrochelatase-Iprotein (NCBI GI NO. 113631036).

SEQ ID NO:50 is the amino acid sequence of a Vitis viniferaferrochelatase-I protein (NCBI GI NO. 147818793).

SEQ ID NO:51 is the amino acid sequence of a rice ferrochelatase-IIprotein (NCBI GI NO. 115463419).

SEQ ID NO:52 is the amino acid sequence of a Cucumis sativusferrochelatase-II protein (NCBI GI NO. 12082085).

SEQ ID NO:53 is the amino acid sequence of a Nicotiana tabacumferrochelatase-II protein (NCBI GI NO. 15147828).

SEQ ID NO:54 is the amino acid sequence of a Synechocystisferrochelatase protein (NCBI GI NO. 1708186).

SEQ ID NO:55 is the nucleotide acid sequence of plasmid PHP30949, anentry clone containing the maize ferrochelatase-I protein (SEQ IDNO:10).

SEQ ID NO:56 is the nucleotide sequence of the cDNA insert ofcfp5n.pk064.n7 and encodes a maize ferrochelatase-II protein (SEQ IDNO:8). SEQ ID NO:56 differs from the nucleotide sequence ofcfp3n.pk004.f12 (SEQ ID NO:7) in that it contains a 12 base pairinsertion in the 5′-UTR, 8 nucleotides before the ATG start codon.

SEQ ID NO:57 is the amino acid sequence presented in SEQ ID NO:240025 ofUS Patent Publication No. US2004031072.

SEQ ID NO:58 is the amino acid sequence presented in SEQ ID NO:20 ofJapanese Patent Publication No. JP2001190168-A.

SEQ ID NO:59 is the amino acid sequence presented in SEQ ID NO:13029 ofUS Patent Publication No. US2006150283.

SEQ ID NO:60 is the amino acid sequence presented in SEQ ID NO:7745 ofUS Patent Publication No. US2006150283.

SEQ ID NO:61 is the amino acid sequence presented in SEQ ID NO:46156 ofJapanese Patent Publication No. JP2005185101.

SEQ ID NO:62 is the amino acid sequence presented in SEQ ID NO:52154 ofJapanese Patent Publication No. JP2005185101.

SEQ ID NO:63 is the amino acid sequence presented in SEQ ID NO:72746 ofUS Patent Publication No. US2004034888-A1.

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825.

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF EMBODIMENTS

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

“Ferrochelatase” (protoheme ferrolyase; EC 4.99.1.1) is the terminalenzyme of the biosynthetic pathway of heme; it catalyses the chelationof ferrous ion into the protoporphyrin IX ring to form protoheme Inhigher plants there is evidence for two isoforms of this enzyme,ferrochelatase-1 and ferrochelatase-2 (Suzuki et al. 2002 J Biol Chem277:4731-4737). The terms “ferrochelatase-I”, “ferrochelatase-1”,“FeC-I” and “FeC-1” are used interchangeably herein. The terms“ferrochelatase-II”, “ferrochelatase-2”, “FeC-II” and “FeC-2” are usedinterchangeably herein.

An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from acDNA library and therefore is a sequence which has been transcribed. AnEST is typically obtained by a single sequencing pass of a cDNA insert.The sequence of an entire cDNA insert is termed the “Full-InsertSequence” (“FIS”). A “Contig” sequence is a sequence assembled from twoor more sequences that can be selected from, but not limited to, thegroup consisting of an EST, FIS and PCR sequence. A sequence encoding anentire or functional protein is termed a “Complete Gene Sequence”(“CGS”) and can be derived from an FIS or a contig.

“Agronomic characteristic” is a measurable parameter including but notlimited to, greenness, yield, growth rate, biomass, fresh weight atmaturation, dry weight at maturation, fruit yield, seed yield, totalplant nitrogen content, fruit nitrogen content, seed nitrogen content,nitrogen content in a vegetative tissue, total plant free amino acidcontent, fruit free amino acid content, seed free amino acid content,free amino acid content in a vegetative tissue, total plant proteincontent, fruit protein content, seed protein content, protein content ina vegetative tissue, drought tolerance, nitrogen uptake, root lodging,harvest index, stalk lodging, plant height, ear height and ear length.

“Transgenic” refers to any cell, cell line, callus, tissue, plant partor plant, the genome of which has been altered by the presence of aheterologous nucleic acid, such as a recombinant DNA construct,including those initial transgenic events as well as those created bysexual crosses or asexual propagation from the initial transgenic event.The term “transgenic” as used herein does not encompass the alterationof the genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. For example, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably and is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from amRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product have been removed.

“Precursor” protein refers to the primary product of translation ofmRNA; i.e., with pre- and pro-peptides still present. Pre- andpro-peptides may be and are not limited to intracellular localizationsignals.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature.

The terms “entry clone” and “entry vector” are used interchangeablyherein.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to,promoters, translation leader sequences, introns, and polyadenylationrecognition sequences. The terms “regulatory sequence” and “regulatoryelement” are used interchangeably herein.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably, and refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” refers to a promoter whose activityis determined by developmental events.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

“Allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome. When the alleles present at a given locus on apair of homologous chromosomes in a diploid plant are the same thatplant is homozygous at that locus. If the alleles present at a givenlocus on a pair of homologous chromosomes in a diploid plant differ thatplant is heterozygous at that locus. If a transgene is present on one ofa pair of homologous chromosomes in a diploid plant that plant ishemizygous at that locus.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the Megalign® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal V method of alignment(Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters(GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments and calculation of percent identity of protein sequencesusing the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAPPENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

Turning now to the embodiments:

Embodiments include isolated polynucleotides and polypeptides,recombinant DNA constructs useful for conferring drought tolerance,compositions (such as plants or seeds) comprising these recombinant DNAconstructs, and methods utilizing these recombinant DNA constructs.

Isolated Polynucleotides and Polypeptides:

The present invention includes the following isolated polynucleotidesand polypeptides:

An isolated polynucleotide comprising: (i) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:4, 6, 8,10, 12, 14, 16, 18, 24, 26, 28 or 30; or (ii) a full complement of thenucleic acid sequence of (i), wherein the full complement and thenucleic acid sequence of (i) consist of the same number of nucleotidesand are 100% complementary. Any of the foregoing isolatedpolynucleotides may be utilized in any recombinant DNA constructs(including suppression DNA constructs) of the present invention. Thepolypeptide is preferably a ferrochelatase.

An isolated polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:4, 6, 8,10, 12, 14, 16, 18, 24, 26, 28 or 30. The polypeptide is preferably aferrochelatase.

An isolated polynucleotide comprising (i) a nucleic acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ IDNO:3, 5, 7, 9, 11, 13, 15, 17, 23, 25, 27 or 29; or (ii) a fullcomplement of the nucleic acid sequence of (i). Any of the foregoingisolated polynucleotides may be utilized in any recombinant DNAconstructs (including suppression DNA constructs) of the presentinvention. The isolated polynucleotide preferably encodes aferrochelatase.

Recombinant DNA Constructs and Suppression DNA Constructs:

In one aspect, the present invention includes recombinant DNA constructs(including suppression DNA constructs).

In one embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one regulatory sequence(e.g., a promoter functional in a plant), wherein the polynucleotidecomprises (i) a nucleic acid sequence encoding an amino acid sequence ofat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; or(ii) a full complement of the nucleic acid sequence of (i).

In another embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one regulatory sequence(e.g., a promoter functional in a plant), wherein said polynucleotidecomprises (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal Vmethod of alignment, when compared to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27 or 29; or (ii) a full complement of thenucleic acid sequence of (i).

FIGS. 10A-10C show the multiple alignment of the amino acid sequences ofthe ferrochelatases of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 48, 49and 50. The multiple alignment of the sequences was performed using theMegalign® program of the LASERGENE® bioinformatics computing suite(DNASTAR® Inc., Madison, Wis.); in particular, using the Clustal Vmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe multiple alignment default parameters of GAP PENALTY=10 and GAPLENGTH PENALTY=10, and the pairwise alignment default parameters ofKTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

FIG. 11 shows the percent sequence identity and the divergence valuesfor each pair of amino acids sequences displayed in FIGS. 10A-10C.

FIGS. 12A-12C show the multiple alignment of the amino acid sequences ofthe ferrochelatases of SEQ ID NO:20, 22, 24, 26, 28, 30, 51, 52, 53 and54. The multiple alignment of the sequences was performed using theMegalign® program of the LASERGENE® bioinformatics computing suite(DNASTAR® Inc., Madison, Wis.); in particular, using the Clustal Vmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe multiple alignment default parameters of GAP PENALTY=10 and GAPLENGTH PENALTY=10, and the pairwise alignment default parameters ofKTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

FIG. 13 shows the percent sequence identity and the divergence valuesfor each pair of amino acids sequences displayed in FIGS. 12A-12C.

In another embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one regulatory sequence(e.g., a promoter functional in a plant), wherein said polynucleotideencodes a ferrochelatase. The ferrochelatase may be from Arabidopsisthaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja andGlycine tomentella.

In another aspect, the present invention includes suppression DNAconstructs.

A suppression DNA construct may comprise at least one regulatorysequence (e.g., a promoter functional in a plant) operably linked to (a)all or part of: (i) a nucleic acid sequence encoding a polypeptidehaving an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity, based on the Clustal V method ofalignment, when compared to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28 or 30, or (ii) a full complement of the nucleic acidsequence of (a)(i); or (b) a region derived from all or part of a sensestrand or antisense strand of a target gene of interest, said regionhaving a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity, based on the Clustal V method ofalignment, when compared to said all or part of a sense strand orantisense strand from which said region is derived, and wherein saidtarget gene of interest encodes a ferrochelatase; or (c) all or part of:(i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity, based on the Clustal V method ofalignment, when compared to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27 or 29, or (ii) a full complement of the nucleic acidsequence of (c)(i). The suppression DNA construct may comprise acosuppression construct, antisense construct, viral-suppressionconstruct, hairpin suppression construct, stem-loop suppressionconstruct, double-stranded RNA-producing construct, RNAi construct, orsmall RNA construct (e.g., an siRNA construct or an miRNA construct).

It is understood, as those skilled in the art will appreciate, that theinvention encompasses more than the specific exemplary sequences.Alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not affectthe functional properties of the encoded polypeptide, are well known inthe art. For example, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another lesshydrophobic residue, such as glycine, or a more hydrophobic residue,such as valine, leucine, or isoleucine. Similarly, changes which resultin substitution of one negatively charged residue for another, such asaspartic acid for glutamic acid, or one positively charged residue foranother, such as lysine for arginine, can also be expected to produce afunctionally equivalent product. Nucleotide changes which result inalteration of the N-terminal and C-terminal portions of the polypeptidemolecule would also not be expected to alter the activity of thepolypeptide. Each of the proposed modifications is well within theroutine skill in the art, as is determination of retention of biologicalactivity of the encoded products.

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The terms“suppression”, “suppressing” and “silencing”, used interchangeablyherein, include lowering, reducing, declining, decreasing, inhibiting,eliminating or preventing. “Silencing” or “gene silencing” does notspecify mechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches, and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target gene orgene product. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target isolated nucleic acid fragment(U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA maybe with any part of the specific gene transcript, i.e., at the 5′non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target gene or geneproduct. “Sense” RNA refers to RNA transcript that includes the mRNA andcan be translated into protein within a cell or in vitro. Cosuppressionconstructs in plants have been previously designed by focusing onoverexpression of a nucleic acid sequence having homology to a nativemRNA, in the sense orientation, which results in the reduction of allRNA having homology to the overexpressed sequence (see Vaucheret et al.,Plant J. 16:651-659 (1998); and Gura, Nature 404:804-808 (2000)).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication No.WO 98/36083 published on Aug. 20, 1998).

Previously described is the use of “hairpin” structures that incorporateall, or part, of an mRNA encoding sequence in a complementaryorientation that results in a potential “stem-loop” structure for theexpressed RNA (PCT Publication No. WO 99/53050 published on Oct. 21,1999). In this case the stem is formed by polynucleotides correspondingto the gene of interest inserted in either sense or anti-senseorientation with respect to the promoter and the loop is formed by somepolynucleotides of the gene of interest, which do not have a complementin the construct. This increases the frequency of cosuppression orsilencing in the recovered transgenic plants. For review of hairpinsuppression see Wesley, S. V. et al. (2003) Methods in MolecularBiology, Plant Functional Genomics: Methods and Protocols 236:273-286.

A construct where the stem is formed by at least 30 nucleotides from agene to be suppressed and the loop is formed by a random nucleotidesequence has also effectively been used for suppression (PCT PublicationNo. WO 99/61632 published on Dec. 2, 1999).

The use of poly-T and poly-A sequences to generate the stem in thestem-loop structure has also been described (PCT Publication No. WO02/00894 published Jan. 3, 2002).

Yet another variation includes using synthetic repeats to promoteformation of a stem in the stem-loop structure. Transgenic organismsprepared with such recombinant DNA fragments have been shown to havereduced levels of the protein encoded by the nucleotide fragment formingthe loop as described in PCT Publication No. WO 02/00904, published Jan.3, 2002.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., Nature 391:806 (1998)). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., Trends Genet.15:358 (1999)). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA of viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., Nature 409:363 (2001)).Short interfering RNAs derived from dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes (Elbashir et al., Genes Dev. 15:188 (2001)). Dicer has alsobeen implicated in the excision of 21- and 22-nucleotide small temporalRNAs (stRNAs) from precursor RNA of conserved structure that areimplicated in translational control (Hutvagner et al., Science 293:834(2001)). The RNAi response also features an endonuclease complex,commonly referred to as an RNA-induced silencing complex (RISC), whichmediates cleavage of single-stranded RNA having sequence complementarityto the antisense strand of the siRNA duplex. Cleavage of the target RNAtakes place in the middle of the region complementary to the antisensestrand of the siRNA duplex. In addition, RNA interference can alsoinvolve small RNA (e.g., miRNA) mediated gene silencing, presumablythrough cellular mechanisms that regulate chromatin structure andthereby prevent transcription of target gene sequences (see, e.g.,Allshire, Science 297:1818-1819 (2002); Volpe et al., Science297:1833-1837 (2002); Jenuwein, Science 297:2215-2218 (2002); and Hallet al., Science 297:2232-2237 (2002)). As such, miRNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al. (Nature391:806 (1998)) were the first to observe RNAi in Caenorhabditiselegans. Wianny and Goetz (Nature Cell Biol. 2:70 (1999)) describe RNAimediated by dsRNA in mouse embryos. Hammond et al. (Nature 404:293(2000)) describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., (Nature 411:494 (2001)) describe RNAi induced byintroduction of duplexes of synthetic 21-nucleotide RNAs in culturedmammalian cells including human embryonic kidney and HeLa cells.

Small RNAs play an important role in controlling gene expression.Regulation of many developmental processes, including flowering, iscontrolled by small RNAs. It is now possible to engineer changes in geneexpression of plant genes by using transgenic constructs which producesmall RNAs in the plant.

Small RNAs appear to function by base-pairing to complementary RNA orDNA target sequences. When bound to RNA, small RNAs trigger either RNAcleavage or translational inhibition of the target sequence. When boundto DNA target sequences, it is thought that small RNAs can mediate DNAmethylation of the target sequence. The consequence of these events,regardless of the specific mechanism, is that gene expression isinhibited.

It is thought that sequence complementarity between small RNAs and theirRNA targets helps to determine which mechanism, RNA cleavage ortranslational inhibition, is employed. It is believed that siRNAs whichare perfectly complementary with their targets, work by RNA cleavage.Some miRNAs have perfect or near-perfect complementarity with theirtargets, and RNA cleavage has been demonstrated for at least a few ofthese miRNAs. Other miRNAs have several mismatches with their targets,and apparently inhibit their targets at the translational level. Again,without being held to a particular theory on the mechanism of action, ageneral rule is emerging that perfect or near-perfect complementaritycauses RNA cleavage, whereas translational inhibition is favored whenthe miRNA/target duplex contains many mismatches. The apparent exceptionto this is microRNA 172 (miR172) in plants. One of the targets of miR172is APETALA2 (AP2), and although miR172 shares near-perfectcomplementarity with AP2 it appears to cause translational inhibition ofAP2 rather than RNA cleavage.

MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about 24nucleotides (nt) in length that have been identified in both animals andplants (Lagos-Quintana et al., Science 294:853-858 (2001),Lagos-Quintana et al., Curr. Biol. 12:735-739 (2002); Lau et al.,Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001);Llave et al., Plant Cell 14:1605-1619 (2002); Mourelatos et al., Genes.Dev. 16:720-728 (2002); Park et al., Curr. Biol. 12:1484-1495 (2002);Reinhart et al., Genes. Dev. 16:1616-1626 (2002)). They are processedfrom longer precursor transcripts that range in size from approximately70 to 200 nt, and these precursor transcripts have the ability to formstable hairpin structures. In animals, the enzyme involved in processingmiRNA precursors is called dicer, an RNAse III-like protein (Grishok etal., Cell 106:23-34 (2001); Hutvagner et al., Science 293:834-838(2001); Ketting et al., Genes. Dev. 15:2654-2659 (2001)). Plants alsohave a dicer-like enzyme, DCL1 (previously named CARPEL FACTORY/SHORTINTEGUMENTS1/SUSPENSOR1), and recent evidence indicates that it, likedicer, is involved in processing the hairpin precursors to generatemature miRNAs (Park et al., Curr. Biol. 12:1484-1495 (2002); Reinhart etal., Genes Dev. 16:1616-1626 (2002)). Furthermore, it is becoming clearfrom recent work that at least some miRNA hairpin precursors originateas longer polyadenylated transcripts, and several different miRNAs andassociated hairpins can be present in a single transcript(Lagos-Quintana et al., Science 294:853-858 (2001); Lee et al., EMBO J.21:4663-4670 (2002)). Recent work has also examined the selection of themiRNA strand from the dsRNA product arising from processing of thehairpin by DICER (Schwartz et al., Cell 115:199-208 (2003)). It appearsthat the stability (i.e. G:C versus A:U content, and/or mismatches) ofthe two ends of the processed dsRNA affects the strand selection, withthe low stability end being easier to unwind by a helicase activity. The5′ end strand at the low stability end is incorporated into the RISCcomplex, while the other strand is degraded.

MicroRNAs (miRNAs) appear to regulate target genes by binding tocomplementary sequences located in the transcripts produced by thesegenes. In the case of lin-4 and let-7, the target sites are located inthe 3′ UTRs of the target mRNAs (Lee et al., Cell 75:843-854 (1993);Wightman et al., Cell 75:855-862 (1993); Reinhart et al., Nature403:901-906 (2000); Slack et al., Mol. Cell. 5:659-669 (2000)), andthere are several mismatches between the lin-4 and let-7 miRNAs andtheir target sites. Binding of the lin-4 or let-7 miRNA appears to causedownregulation of steady-state levels of the protein encoded by thetarget mRNA without affecting the transcript itself (Olsen and Ambros,Dev. Biol. 216:671-680 (1999)). On the other hand, recent evidencesuggests that miRNAs can in some cases cause specific RNA cleavage ofthe target transcript within the target site, and this cleavage stepappears to require 100% complementarity between the miRNA and the targettranscript (Hutvagner and Zamore, Science 297:2056-2060 (2002); Llave etal., Plant Cell 14:1605-1619 (2002)). It seems likely that miRNAs canenter at least two pathways of target gene regulation: (1) proteindownregulation when target complementarity is <100%; and (2) RNAcleavage when target complementarity is 100%. MicroRNAs entering the RNAcleavage pathway are analogous to the 21-25 nt short interfering RNAs(siRNAs) generated during RNA interference (RNAi) in animals andposttranscriptional gene silencing (PTGS) in plants, and likely areincorporated into an RNA-induced silencing complex (RISC) that issimilar or identical to that seen for RNAi.

Identifying the targets of miRNAs with bioinformatics has not beensuccessful in animals, and this is probably due to the fact that animalmiRNAs have a low degree of complementarity with their targets. On theother hand, bioinformatic approaches have been successfully used topredict targets for plant miRNAs (Llave et al., Plant Cell 14:1605-1619(2002); Park et al., Curr. Biol. 12:1484-1495 (2002); Rhoades et al.,Cell 110:513-520 (2002)), and thus it appears that plant miRNAs havehigher overall complementarity with their putative targets than doanimal miRNAs. Most of these predicted target transcripts of plantmiRNAs encode members of transcription factor families implicated inplant developmental patterning or cell differentiation.

Regulatory Sequences:

A recombinant DNA construct (including a suppression DNA construct) ofthe present invention may comprise at least one regulatory sequence.

A regulatory sequence may be a promoter.

A number of promoters can be used in recombinant DNA constructs of thepresent invention. The promoters can be selected based on the desiredoutcome, and may include constitutive, tissue-specific, inducible, orother promoters for expression in the host organism.

High level, constitutive expression of the candidate gene under controlof the 35S or UBI promoter may have pleiotropic effects, althoughcandidate gene efficacy may be estimated when driven by a constitutivepromoter. Use of tissue-specific and/or stress-specific promoters mayeliminate undesirable effects but retain the ability to enhance droughttolerance. This effect has been observed in Arabidopsis (Kasuga et al.(1999) Nature Biotechnol. 17:287-91).

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812(1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

In choosing a promoter to use in the methods of the invention, it may bedesirable to use a tissue-specific or developmentally regulatedpromoter.

A tissue-specific or developmentally regulated promoter is a DNAsequence which regulates the expression of a DNA sequence selectively inthe cells/tissues of a plant critical to tassel development, seed set,or both, and limits the expression of such a DNA sequence to the periodof tassel development or seed maturation in the plant. Any identifiablepromoter may be used in the methods of the present invention whichcauses the desired temporal and spatial expression.

Promoters which are seed or embryo-specific and may be useful in theinvention include soybean Kunitz trypsin inhibitor (Kti3, Jofuku andGoldberg, Plant Cell 1:1079-1093 (1989)), patatin (potato tubers)(Rocha-Sosa, M., et al. (1989) EMBO J. 8:23-29), convicilin, vicilin,and legumin (pea cotyledons) (Rerie, W. G., et al. (1991) Mol. Gen.Genet. 259:149-157; Newbigin, E. J., et al. (1990) Planta 180:461-470;Higgins, T. J. V., et al. (1988) Plant. Mol. Biol. 11:683-695), zein(maize endosperm) (Schemthaner, J. P., et al. (1988) EMBO J.7:1249-1255), phaseolin (bean cotyledon) (Segupta-Gopalan, C., et al.(1985) Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324), phytohemagglutinin(bean cotyledon) (Voelker, T. et al. (1987) EMBO J. 6:3571-3577),B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L, et al. (1988)EMBO J. 7:297-302), glutelin (rice endosperm), hordein (barleyendosperm) (Marris, C., et al. (1988) Plant Mol. Biol. 10:359-366),glutenin and gliadin (wheat endosperm) (Colot, V., et al. (1987) EMBO J.6:3559-3564), and sporamin (sweet potato tuberous root) (Hattori, T., etal. (1990) Plant Mol. Biol. 14:595-604). Promoters of seed-specificgenes operably linked to heterologous coding regions in chimeric geneconstructions maintain their temporal and spatial expression pattern intransgenic plants. Such examples include Arabidopsis thaliana 2S seedstorage protein gene promoter to express enkephalin peptides inArabidopsis and Brassica napus seeds (Vanderkerckhove et al.,Bio/Technology 7:L929-932 (1989)), bean lectin and bean beta-phaseolinpromoters to express luciferase (Riggs et al., Plant Sci. 63:47-57(1989)), and wheat glutenin promoters to express chloramphenicol acetyltransferase (Colot et al., EMBO J. 6:3559-3564 (1987)).

Inducible promoters selectively express an operably linked DNA sequencein response to the presence of an endogenous or exogenous stimulus, forexample by chemical compounds (chemical inducers) or in response toenvironmental, hormonal, chemical, and/or developmental signals.Inducible or regulated promoters include, for example, promotersregulated by light, heat, stress, flooding or drought, phytohormones,wounding, or chemicals such as ethanol, jasmonate, salicylic acid, orsafeners.

Promoters for use in the current invention include the following: 1) thestress-inducible RD29A promoter (Kasuga et al. (1999) Nature Biotechnol.17:287-91); 2) the barley promoter, B22E; expression of B22E is specificto the pedicel in developing maize kernels (“Primary Structure of aNovel Barley Gene Differentially Expressed in Immature Aleurone Layers”.Klemsdal, S. S. et al., Mol. Gen. Genet. 228(½):9-16 (1991)); and 3)maize promoter, Zag2 (“Identification and molecular characterization ofZAG1, the maize homolog of the Arabidopsis floral homeotic geneAGAMOUS”, Schmidt, R. J. et al., Plant Cell 5(7):729-737 (1993);“Structural characterization, chromosomal localization and phylogeneticevaluation of two pairs of AGAMOUS-like MADS-box genes from maize”,Theissen et al. Gene 156(2):155-166 (1995); NCBI GenBank Accession No.X80206)). Zag2 transcripts can be detected 5 days prior to pollinationto 7 to 8 days after pollination (“DAP”), and directs expression in thecarpel of developing female inflorescences and CimI which is specific tothe nucleus of developing maize kernels. CimI transcript is detected 4to 5 days before pollination to 6 to 8 DAP. Other useful promotersinclude any promoter which can be derived from a gene whose expressionis maternally associated with developing female florets.

Additional promoters for regulating the expression of the nucleotidesequences of the present invention in plants are stalk-specificpromoters. Such stalk-specific promoters include the alfalfa S2Apromoter (GenBank Accession No. EF030816; Abrahams et al., Plant Mol.Biol. 27:513-528 (1995)) and S2B promoter (GenBank Accession No.EF030817) and the like, herein incorporated by reference.

Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of some variation may have identical promoter activity.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. Newpromoters of various types useful in plant cells are constantly beingdiscovered; numerous examples may be found in the compilation byOkamuro, J. K., and Goldberg, R. B., Biochemistry of Plants 15:1-82(1989).

Promoters for use in the current invention may include: RIP2, mLIP15,ZmCOR1, Rab17, CaMV 35S, RD29A, B22E, Zag2, SAM synthetase, ubiquitin,CaMV 19S, nos, Adh, sucrose synthase, R-allele, the vascular tissuepreferred promoters S2A (Genbank accession number EF030816) and S2B(Genbank accession number EF030817), and the constitutive promoter GOS2from Zea mays. Other promoters include root preferred promoters, such asthe maize NAS2 promoter, the maize Cyclo promoter (US 2006/0156439,published Jul. 13, 2006), the maize ROOTMET2 promoter (WO05063998,published Jul. 14, 2005), the CR1BIO promoter (WO06055487, published May26, 2006), the CRWAQ81 (WO05035770, published Apr. 21, 2005) and themaize ZRP2.47 promoter (NCBI accession number: U38790; GI No. 1063664),

Recombinant DNA constructs of the present invention may also includeother regulatory sequences, including but not limited to, translationleader sequences, introns, and polyadenylation recognition sequences. Inanother embodiment of the present invention, a recombinant DNA constructof the present invention further comprises an enhancer or silencer.

An intron sequence can be added to the 5′ untranslated region, theprotein-coding region or the 3′ untranslated region to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold. Buchman and Berg,Mol. Cell. Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987). Such intron enhancement of gene expression istypically greatest when placed near the 5′ end of the transcriptionunit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1intron are known in the art. See generally, The Maize Handbook, Chapter116, Freeling and Walbot, Eds., Springer, New York (1994).

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or from a non-plant eukaryotic gene.

A translation leader sequence is a DNA sequence located between thepromoter sequence of a gene and the coding sequence. The translationleader sequence is present in the fully processed mRNA upstream of thetranslation start sequence. The translation leader sequence may affectprocessing of the primary transcript to mRNA, mRNA stability ortranslation efficiency. Examples of translation leader sequences havebeen described (Turner, R. and Foster, G. D. (1995) MolecularBiotechnology 3:225).

Any plant can be selected for the identification of regulatory sequencesand ferrochelatase genes to be used in recombinant DNA constructs of thepresent invention. Examples of suitable plant targets for the isolationof genes and regulatory sequences would include but are not limited toalfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus,avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli,brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava,castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus,clementines, clover, coconut, coffee, corn, cotton, cranberry, cucumber,Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs,garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce,leeks, lemon, lime, Loblolly pine, linseed, mango, melon, mushroom,nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion,orange, an ornamental plant, palm, papaya, parsley, parsnip, pea, peach,peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum,pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio,radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweetpotato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf,turnip, a vine, watermelon, wheat, yams, and zucchini.

Compositions:

A composition of the present invention is a plant comprising in itsgenome any of the recombinant DNA constructs (including any of thesuppression DNA constructs) of the present invention (such as any of theconstructs discussed above). Compositions also include any progeny ofthe plant, and any seed obtained from the plant or its progeny, whereinthe progeny or seed comprises within its genome the recombinant DNAconstruct (or suppression DNA construct). Progeny includes subsequentgenerations obtained by self-pollination or out-crossing of a plant.Progeny also includes hybrids and inbreds.

In hybrid seed propagated crops, mature transgenic plants can beself-pollinated to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced recombinant DNA construct(or suppression DNA construct). These seeds can be grown to produceplants that would exhibit an altered agronomic characteristic (e.g., anincreased agronomic characteristic optionally under water limitingconditions), or used in a breeding program to produce hybrid seed, whichcan be grown to produce plants that would exhibit such an alteredagronomic characteristic. The seeds may be maize seeds.

The plant may be a monocotyledonous or dicotyledonous plant, forexample, a maize or soybean plant, such as a maize hybrid plant or amaize inbred plant. The plant may also be sunflower, sorghum, canola,wheat, alfalfa, cotton, rice, barley or millet.

The recombinant DNA construct may be stably integrated into the genomeof the plant.

Particularly embodiments include but are not limited to the following:

1. A plant (for example, a maize or soybean plant) comprising in itsgenome a recombinant DNA construct comprising a polynucleotide operablylinked to at least one regulatory sequence, wherein said polynucleotideencodes a polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, and wherein said plantexhibits increased drought tolerance when compared to a control plantnot comprising said recombinant DNA construct. The plant may furtherexhibit an alteration of at least one agronomic characteristic whencompared to the control plant.

2. A plant (for example, a maize or soybean plant) comprising in itsgenome a recombinant DNA construct comprising a polynucleotide operablylinked to at least one regulatory sequence, wherein said polynucleotideencodes a ferrochelatase, and wherein said plant exhibits increaseddrought tolerance when compared to a control plant not comprising saidrecombinant DNA construct. The plant may further exhibit an alterationof at least one agronomic characteristic when compared to the controlplant.

3. A plant (for example, a maize or soybean plant) comprising in itsgenome a recombinant DNA construct comprising a polynucleotide operablylinked to at least one regulatory sequence, wherein said polynucleotideencodes a ferrochelatase, and wherein said plant exhibits an alterationof at least one agronomic characteristic when compared to a controlplant not comprising said recombinant DNA construct.

4. A plant (for example, a maize or soybean plant) comprising in itsgenome a recombinant DNA construct comprising a polynucleotide operablylinked to at least one regulatory element, wherein said polynucleotideencodes a polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, and wherein said plantexhibits an alteration of at least one agronomic characteristic whencompared to a control plant not comprising said recombinant DNAconstruct.

5. A plant (for example, a maize or soybean plant) comprising in itsgenome a suppression DNA construct comprising at least one regulatoryelement operably linked to a region derived from all or part of a sensestrand or antisense strand of a target gene of interest, said regionhaving a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity, based on the Clustal V method ofalignment, when compared to said all or part of a sense strand orantisense strand from which said region is derived, and wherein saidtarget gene of interest encodes a ferrochelatase, and wherein said plantexhibits an alteration of at least one agronomic characteristic whencompared to a control plant not comprising said suppression DNAconstruct.

6. A plant (for example, a maize or soybean plant) comprising in itsgenome a suppression DNA construct comprising at least one regulatoryelement operably linked to all or part of (a) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, or (b) a fullcomplement of the nucleic acid sequence of (a), and wherein said plantexhibits an alteration of at least one agronomic characteristic whencompared to a control plant not comprising said suppression DNAconstruct.

7. Any progeny of the above plants in embodiments 1-6, any seeds of theabove plants in embodiments 1-6, any seeds of progeny of the aboveplants in embodiments 1-6, and cells from any of the above plants inembodiments 1-6 and progeny thereof.

In any of the foregoing embodiments 1-7 or any other embodiments of thepresent invention, the ferrochelatase may be from Arabidopsis thaliana,Zea mays, Glycine max, Glycine tabacina, Glycine soja or Glycinetomentella.

In any of the foregoing embodiments 1-7 or any other embodiments of thepresent invention, the recombinant DNA construct (or suppression DNAconstruct) may comprise at least a promoter functional in a plant as aregulatory sequence.

In any of the foregoing embodiments 1-7 or any other embodiments of thepresent invention, the alteration of at least one agronomiccharacteristic is either an increase or decrease.

In any of the foregoing embodiments 1-7 or any other embodiments of thepresent invention, the at least one agronomic characteristic may beselected from the group consisting of greenness, yield, growth rate,biomass, fresh weight at maturation, dry weight at maturation, fruityield, seed yield, total plant nitrogen content, fruit nitrogen content,seed nitrogen content, nitrogen content in a vegetative tissue, totalplant free amino acid content, fruit free amino acid content, seed freeamino acid content, free amino acid content in a vegetative tissue,total plant protein content, fruit protein content, seed proteincontent, protein content in a vegetative tissue, drought tolerance,nitrogen uptake, root lodging, harvest index, stalk lodging, plantheight, ear height and ear length. For example, the alteration of atleast one agronomic characteristic may be an increase in yield,greenness or biomass.

In any of the foregoing embodiments 1-7 or any other embodiments of thepresent invention, the plant may exhibit the alteration of at least oneagronomic characteristic when compared, under water limiting conditions,to a control plant not comprising said recombinant DNA construct (orsaid suppression DNA construct).

“Drought” refers to a decrease in water availability to a plant that,especially when prolonged, can cause damage to the plant or prevent itssuccessful growth (e.g., limiting plant growth or seed yield).

“Drought tolerance” is a trait of a plant to survive under droughtconditions over prolonged periods of time without exhibiting substantialphysiological or physical deterioration.

“Increased drought tolerance” of a plant is measured relative to areference or control plant, and is a trait of the plant to survive underdrought conditions over prolonged periods of time, without exhibitingthe same degree of physiological or physical deterioration relative tothe reference or control plant grown under similar drought conditions.Typically, when a transgenic plant comprising a recombinant DNAconstruct or suppression DNA construct in its genome exhibits increaseddrought tolerance relative to a reference or control plant, thereference or control plant does not comprise in its genome therecombinant DNA construct or suppression DNA construct.

One of ordinary skill in the art is familiar with protocols forsimulating drought conditions and for evaluating drought tolerance ofplants that have been subjected to simulated or naturally-occurringdrought conditions. For example, one can simulate drought conditions bygiving plants less water than normally required or no water over aperiod of time, and one can evaluate drought tolerance by looking fordifferences in physiological and/or physical condition, including (butnot limited to) vigor, growth, size, or root length, or in particular,leaf color or leaf area size. Other techniques for evaluating droughttolerance include measuring chlorophyll fluorescence, photosyntheticrates and gas exchange rates.

A drought stress experiment may involve a chronic stress (i.e., slow drydown) and/or may involve two acute stresses (i.e., abrupt removal ofwater) separated by a day or two of recovery. Chronic stress may last8-10 days. Acute stress may last 3-5 days. The following variables maybe measured during drought stress and well watered treatments oftransgenic plants and relevant control plants:

The variable “% area chg_start chronic−acute2” is a measure of thepercent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and the day of thesecond acute stress

The variable “% area chg_start chronic−end chronic” is a measure of thepercent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and the last day ofchronic stress

The variable “% area chg_start chronic−harvest” is a measure of thepercent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and the day of harvest

The variable “% area chg_start chronic−recovery24 hr” is a measure ofthe percent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and 24 hrs into therecovery (24 hrs after acute stress 2)

The variable “psii_acute1” is a measure of Photosystem II (PSII)efficiency at the end of the first acute stress period. It provides anestimate of the efficiency at which light is absorbed by PSII antennaeand is directly related to carbon dioxide assimilation within the leaf.

The variable “psii_acute2” is a measure of Photosystem II (PSII)efficiency at the end of the second acute stress period. It provides anestimate of the efficiency at which light is absorbed by PSII antennaeand is directly related to carbon dioxide assimilation within the leaf.

The variable “fv/fm_acute1” is a measure of the optimum quantum yield(Fv/Fm) at the end of the first acute stress−(variable fluorescencedifference between the maximum and minimum fluorescence/maximumfluorescence)

The variable “fv/fm_acute2” is a measure of the optimum quantum yield(Fv/Fm) at the end of the second acute stress−(variable fluorescencedifference between the maximum and minimum fluorescence/maximumfluorescence)

The variable “leaf rolling_harvest” is a measure of the ratio of topimage to side image on the day of harvest.

The variable “leaf rolling_recovery24 hr” is a measure of the ratio oftop image to side image 24 hours into the recovery.

The variable “Specific Growth Rate (SGR)” represents the change in totalplant surface area (as measured by Lemna Tec Instrument) over a singleday (Y(t)=Y0*e^(r*t)). Y(t)=Y0*e^(r*t) is equivalent to % change in Y/Δt where the individual terms are as follows: Y(t)=Total surface area att; Y0=Initial total surface area (estimated); r=Specific Growth Rateday⁻¹, and t=Days After Planting (“DAP”)

The variable “shoot dry weight” is a measure of the shoot weight 96hours after being placed into a 104° C. oven

The variable “shoot fresh weight” is a measure of the shoot weightimmediately after being cut from the plant

The Examples below describe some representative protocols and techniquesfor simulating drought conditions and/or evaluating drought tolerance.

One can also evaluate drought tolerance by the ability of a plant tomaintain sufficient yield (at least 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% yield) in field testing under simulated ornaturally-occurring drought conditions (e.g., by measuring forsubstantially equivalent yield under drought conditions compared tonon-drought conditions, or by measuring for less yield loss underdrought conditions compared to a control or reference plant).

One of ordinary skill in the art would readily recognize a suitablecontrol or reference plant to be utilized when assessing or measuring anagronomic characteristic or phenotype of a transgenic plant in anyembodiment of the present invention in which a control plant is utilized(e.g., compositions or methods as described herein). For example, by wayof non-limiting illustrations:

1. Progeny of a transformed plant which is hemizygous with respect to arecombinant DNA construct (or suppression DNA construct), such that theprogeny are segregating into plants either comprising or not comprisingthe recombinant DNA construct (or suppression DNA construct): theprogeny comprising the recombinant DNA construct (or suppression DNAconstruct) would be typically measured relative to the progeny notcomprising the recombinant DNA construct (or suppression DNA construct)(i.e., the progeny not comprising the recombinant DNA construct (or thesuppression DNA construct) is the control or reference plant).

2. Introgression of a recombinant DNA construct (or suppression DNAconstruct) into an inbred line, such as in maize, or into a variety,such as in soybean: the introgressed line would typically be measuredrelative to the parent inbred or variety line (i.e., the parent inbredor variety line is the control or reference plant).

3. Two hybrid lines, where the first hybrid line is produced from twoparent inbred lines, and the second hybrid line is produced from thesame two parent inbred lines except that one of the parent inbred linescontains a recombinant DNA construct (or suppression DNA construct): thesecond hybrid line would typically be measured relative to the firsthybrid line (i.e., the first hybrid line is the control or referenceplant).

4. A plant comprising a recombinant DNA construct (or suppression DNAconstruct): the plant may be assessed or measured relative to a controlplant not comprising the recombinant DNA construct (or suppression DNAconstruct) but otherwise having a comparable genetic background to theplant (e.g., sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity of nuclear genetic material comparedto the plant comprising the recombinant DNA construct (or suppressionDNA construct)). There are many laboratory-based techniques availablefor the analysis, comparison and characterization of plant geneticbackgrounds; among these are Isozyme Electrophoresis, RestrictionFragment Length Polymorphisms (RFLPs), Randomly Amplified PolymorphicDNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Amplified Fragment Length Polymorphisms (AFLP®s), andSimple Sequence Repeats (SSRs) which are also referred to asMicrosatellites.

Furthermore, one of ordinary skill in the art would readily recognizethat a suitable control or reference plant to be utilized when assessingor measuring an agronomic characteristic or phenotype of a transgenicplant would not include a plant that had been previously selected, viamutagenesis or transformation, for the desired agronomic characteristicor phenotype.

Methods:

Methods include but are not limited to methods for increasing droughttolerance in a plant, methods for evaluating drought tolerance in aplant, methods for altering an agronomic characteristic in a plant,methods for determining an alteration of an agronomic characteristic ina plant, and methods for producing seed. The plant may be amonocotyledonous or dicotyledonous plant, for example, a maize orsoybean plant. The plant may also be sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley or millet. The seed is may be a maize orsoybean seed, for example, a maize hybrid seed or maize inbred seed.

Methods include but are not limited to the following:

A method for transforming a cell comprising transforming a cell with anyof the isolated polynucleotides of the present invention. The celltransformed by this method is also included. In particular embodiments,the cell is eukaryotic cell, e.g., a yeast, insect or plant cell, orprokaryotic, e.g., a bacterium.

A method for producing a transgenic plant comprising transforming aplant cell with any of the isolated polynucleotides or recombinant DNAconstructs of the present invention and regenerating a transgenic plantfrom the transformed plant cell. The invention is also directed to thetransgenic plant produced by this method, and transgenic seed obtainedfrom this transgenic plant.

A method for isolating a polypeptide of the invention from a cell orculture medium of the cell, wherein the cell comprises a recombinant DNAconstruct comprising a polynucleotide of the invention operably linkedto at least one regulatory sequence, and wherein the transformed hostcell is grown under conditions that are suitable for expression of therecombinant DNA construct.

A method of altering the level of expression of a polypeptide of theinvention in a host cell comprising: (a) transforming a host cell with arecombinant DNA construct of the present invention; and (b) growing thetransformed host cell under conditions that are suitable for expressionof the recombinant DNA construct wherein expression of the recombinantDNA construct results in production of altered levels of the polypeptideof the invention in the transformed host cell.

A method of increasing drought tolerance in a plant, comprising: (a)introducing into a regenerable plant cell a recombinant DNA constructcomprising a polynucleotide operably linked to at least one regulatorysequence (for example, a promoter functional in a plant), wherein thepolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; and (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct and exhibits increased drought tolerance whencompared to a control plant not comprising the recombinant DNAconstruct. The method may further comprise (c) obtaining a progeny plantderived from the transgenic plant, wherein said progeny plant comprisesin its genome the recombinant DNA construct and exhibits increaseddrought tolerance when compared to a control plant not comprising therecombinant DNA construct.

A method of increasing drought tolerance in a plant, comprising: (a)introducing into a regenerable plant cell a suppression DNA constructcomprising at least one regulatory sequence (for example, a promoterfunctional in a plant) operably linked to all or part of (i) a nucleicacid sequence encoding a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, or (ii) afull complement of the nucleic acid sequence of (a) (i); and (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome thesuppression DNA construct and exhibits increased drought tolerance whencompared to a control plant not comprising the suppression DNAconstruct. The method may further comprise (c) obtaining a progeny plantderived from the transgenic plant, wherein said progeny plant comprisesin its genome the suppression DNA construct and exhibits increaseddrought tolerance when compared to a control plant not comprising thesuppression DNA construct.

A method of increasing drought tolerance in a plant, comprising: (a)introducing into a regenerable plant cell a suppression DNA constructcomprising at least one regulatory sequence (for example, a promoterfunctional in a plant) operably linked to a region derived from all orpart of a sense strand or antisense strand of a target gene of interest,said region having a nucleic acid sequence of at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the ClustalV method of alignment, when compared to said all or part of a sensestrand or antisense strand from which said region is derived, andwherein said target gene of interest encodes a ferrochelatase; and (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome thesuppression DNA construct and exhibits increased drought tolerance whencompared to a control plant not comprising the suppression DNAconstruct. The method may further comprise (c) obtaining a progeny plantderived from the transgenic plant, wherein said progeny plant comprisesin its genome the suppression DNA construct and exhibits increaseddrought tolerance when compared to a control plant not comprising thesuppression DNA construct.

A method of evaluating drought tolerance in a plant, comprising (a)introducing into a regenerable plant cell a recombinant DNA constructcomprising a polynucleotide operably linked to at least on regulatorysequence (for example, a promoter functional in a plant), wherein thepolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct; and (c) evaluating the transgenic plant fordrought tolerance compared to a control plant not comprising therecombinant DNA construct. The method may further comprise (d) obtaininga progeny plant derived from the transgenic plant, wherein the progenyplant comprises in its genome the recombinant DNA construct; and (e)evaluating the progeny plant for drought tolerance compared to a controlplant not comprising the recombinant DNA construct.

A method of evaluating drought tolerance in a plant, comprising (a)introducing into a regenerable plant cell a suppression DNA constructcomprising at least one regulatory sequence (for example, a promoterfunctional in a plant) operably linked to all or part of (i) a nucleicacid sequence encoding a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, or (ii) afull complement of the nucleic acid sequence of (a)(i); (b) regeneratinga transgenic plant from the regenerable plant cell after step (a),wherein the transgenic plant comprises in its genome the suppression DNAconstruct; and (c) evaluating the transgenic plant for drought tolerancecompared to a control plant not comprising the suppression DNAconstruct. The method may further comprise (d) obtaining a progeny plantderived from the transgenic plant, wherein the progeny plant comprisesin its genome the suppression DNA construct; and (e) evaluating theprogeny plant for drought tolerance compared to a control plant notcomprising the suppression DNA construct.

A method of evaluating drought tolerance in a plant, comprising (a)introducing into a regenerable plant cell a suppression DNA constructcomprising at least one regulatory sequence (for example, a promoterfunctional in a plant) operably linked to a region derived from all orpart of a sense strand or antisense strand of a target gene of interest,said region having a nucleic acid sequence of at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the ClustalV method of alignment, when compared to said all or part of a sensestrand or antisense strand from which said region is derived, andwherein said target gene of interest encodes a ferrochelatase; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome thesuppression DNA construct; and (c) evaluating the transgenic plant fordrought tolerance compared to a control plant not comprising thesuppression DNA construct. The method may further comprise (d) obtaininga progeny plant derived from the transgenic plant, wherein the progenyplant comprises in its genome the suppression DNA construct; and (e)evaluating the progeny plant for drought tolerance compared to a controlplant not comprising the suppression DNA construct.

A method of evaluating drought tolerance in a plant, comprising (a)introducing into a regenerable plant cell a recombinant DNA constructcomprising a polynucleotide operably linked to at least one regulatorysequence (for example, a promoter functional in a plant), wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct; (c) obtaining a progeny plant derived fromsaid transgenic plant, wherein the progeny plant comprises in its genomethe recombinant DNA construct; and (d) evaluating the progeny plant fordrought tolerance compared to a control plant not comprising therecombinant DNA construct.

A method of evaluating drought tolerance in a plant, comprising (a)introducing into a regenerable plant cell a suppression DNA constructcomprising at least one regulatory sequence (for example, a promoterfunctional in a plant) operably linked to all or part of (i) a nucleicacid sequence encoding a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, or (ii) afull complement of the nucleic acid sequence of (a)(i); (b) regeneratinga transgenic plant from the regenerable plant cell after step (a),wherein the transgenic plant comprises in its genome the suppression DNAconstruct; (c) obtaining a progeny plant derived from said transgenicplant, wherein the progeny plant comprises in its genome the suppressionDNA construct; and (d) evaluating the progeny plant for droughttolerance compared to a control plant not comprising the suppression DNAconstruct.

A method of evaluating drought tolerance in a plant, comprising (a)introducing into a regenerable plant cell a suppression DNA constructcomprising at least one regulatory sequence (for example, a promoterfunctional in a plant) operably linked to a region derived from all orpart of a sense strand or antisense strand of a target gene of interest,said region having a nucleic acid sequence of at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the ClustalV method of alignment, when compared to said all or part of a sensestrand or antisense strand from which said region is derived, andwherein said target gene of interest encodes a ferrochelatase; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome thesuppression DNA construct; (c) obtaining a progeny plant derived fromthe transgenic plant, wherein the progeny plant comprises in its genomethe suppression DNA construct; and (d) evaluating the progeny plant fordrought tolerance compared to a control plant not comprising thesuppression DNA construct.

A method of determining an alteration of an agronomic characteristic ina plant, comprising (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least on regulatory sequence (for example, a promoter functional in aplant), wherein said polynucleotide encodes a polypeptide having anamino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28or 30; (b) regenerating a transgenic plant from the regenerable plantcell after step (a), wherein the transgenic plant comprises in itsgenome said recombinant DNA construct; and (c) determining whether thetransgenic plant exhibits an alteration in at least one agronomiccharacteristic when compared, optionally under water limitingconditions, to a control plant not comprising the recombinant DNAconstruct. The method may further comprise (d) obtaining a progeny plantderived from the transgenic plant, wherein the progeny plant comprisesin its genome the recombinant DNA construct; and (e) determining whetherthe progeny plant exhibits an alteration in at least one agronomiccharacteristic when compared, optionally under water limitingconditions, to a control plant not comprising the recombinant DNAconstruct.

A method of determining an alteration of an agronomic characteristic ina plant, comprising (a) introducing into a regenerable plant cell asuppression DNA construct comprising at least one regulatory sequence(for example, a promoter functional in a plant) operably linked to allor part of (i) a nucleic acid sequence encoding a polypeptide having anamino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28or 30, or (ii) a full complement of the nucleic acid sequence of (i);(b) regenerating a transgenic plant from the regenerable plant cellafter step (a), wherein the transgenic plant comprises in its genome thesuppression DNA construct; and (c) determining whether the transgenicplant exhibits an alteration in at least one agronomic characteristicwhen compared, optionally under water limiting conditions, to a controlplant not comprising the suppression DNA construct. The method mayfurther comprise (d) obtaining a progeny plant derived from thetransgenic plant, wherein the progeny plant comprises in its genome thesuppression DNA construct; and (e) determining whether the progeny plantexhibits an alteration in at least one agronomic characteristic whencompared, optionally under water limiting conditions, to a control plantnot comprising the suppression DNA construct.

A method of determining an alteration of an agronomic characteristic ina plant, comprising (a) introducing into a regenerable plant cell asuppression DNA construct comprising at least one regulatory sequence(for example, a promoter functional in a plant) operably linked to aregion derived from all or part of a sense strand or antisense strand ofa target gene of interest, said region having a nucleic acid sequence ofat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, based on the Clustal V method of alignment, when compared tosaid all or part of a sense strand or antisense strand from which saidregion is derived, and wherein said target gene of interest encodes aferrochelatase; (b) regenerating a transgenic plant from the regenerableplant cell after step (a), wherein the transgenic plant comprises in itsgenome the suppression DNA construct; and (c) determining whether thetransgenic plant exhibits an alteration in at least one agronomiccharacteristic when compared, optionally under water limitingconditions, to a control plant not comprising the suppression DNAconstruct. The method may further comprise (d) obtaining a progeny plantderived from the transgenic plant, wherein the progeny plant comprisesin its genome the suppression DNA construct; and (e) determining whetherthe progeny plant exhibits an alteration in at least one agronomiccharacteristic when compared, optionally under water limitingconditions, to a control plant not comprising the suppression DNAconstruct.

A method of determining an alteration of an agronomic characteristic ina plant, comprising (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory sequence (for example, a promoter functional ina plant), wherein said polynucleotide encodes a polypeptide having anamino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28or 30; (b) regenerating a transgenic plant from the regenerable plantcell after step (a), wherein the transgenic plant comprises in itsgenome said recombinant DNA construct; (c) obtaining a progeny plantderived from said transgenic plant, wherein the progeny plant comprisesin its genome the recombinant DNA construct; and (d) determining whetherthe progeny plant exhibits an alteration in at least one agronomiccharacteristic when compared, optionally under water limitingconditions, to a control plant not comprising the recombinant DNAconstruct.

A method of determining an alteration of an agronomic characteristic ina plant, comprising (a) introducing into a regenerable plant cell asuppression DNA construct comprising at least one regulatory sequence(for example, a promoter functional in a plant) operably linked to allor part of (i) a nucleic acid sequence encoding a polypeptide having anamino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28or 30, or (ii) a full complement of the nucleic acid sequence of (i);(b) regenerating a transgenic plant from the regenerable plant cellafter step (a), wherein the transgenic plant comprises in its genome thesuppression DNA construct; (c) obtaining a progeny plant derived fromsaid transgenic plant, wherein the progeny plant comprises in its genomethe suppression DNA construct; and (d) determining whether the progenyplant exhibits an alteration in at least one agronomic characteristicwhen compared, optionally under water limiting conditions, to a controlplant not comprising the suppression DNA construct.

A method of determining an alteration of an agronomic characteristic ina plant, comprising (a) introducing into a regenerable plant cell asuppression DNA construct comprising at least one regulatory sequence(for example, a promoter functional in a plant) operably linked to aregion derived from all or part of a sense strand or antisense strand ofa target gene of interest, said region having a nucleic acid sequence ofat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, based on the Clustal V method of alignment, when compared tosaid all or part of a sense strand or antisense strand from which saidregion is derived, and wherein said target gene of interest encodes aferrochelatase; (b) regenerating a transgenic plant from the regenerableplant cell after step (a), wherein the transgenic plant comprises in itsgenome the suppression DNA construct; (c) obtaining a progeny plantderived from said transgenic plant, wherein the progeny plant comprisesin its genome the suppression DNA construct; and (d) determining whetherthe progeny plant exhibits an alteration in at least one agronomiccharacteristic when compared, optionally under water limitingconditions, to a control plant not comprising the suppression DNAconstruct.

A method of producing seed (for example, seed that can be sold as adrought tolerant product offering) comprising any of the precedingmethods, and further comprising obtaining seeds from said progeny plant,wherein said seeds comprise in their genome said recombinant DNAconstruct (or suppression DNA construct).

In any of the preceding methods or any other embodiments of methods ofthe present invention, in said introducing step said regenerable plantcell may comprise a callus cell, an embryogenic callus cell, a gameticcell, a meristematic cell, or a cell of an immature embryo. Theregenerable plant cells may derive from an inbred maize plant.

In any of the preceding methods or any other embodiments of methods ofthe present invention, said regenerating step may comprise thefollowing: (i) culturing said transformed plant cells in a mediacomprising an embryogenic promoting hormone until callus organization isobserved; (ii) transferring said transformed plant cells of step (i) toa first media which includes a tissue organization promoting hormone;and (iii) subculturing said transformed plant cells after step (ii) ontoa second media, to allow for shoot elongation, root development or both.

In any of the preceding methods or any other embodiments of methods ofthe present invention, the at least one agronomic characteristic may beselected from the group consisting of greenness, yield, growth rate,biomass, fresh weight at maturation, dry weight at maturation, fruityield, seed yield, total plant nitrogen content, fruit nitrogen content,seed nitrogen content, nitrogen content in a vegetative tissue, totalplant free amino acid content, fruit free amino acid content, seed freeamino acid content, amino acid content in a vegetative tissue, totalplant protein content, fruit protein content, seed protein content,protein content in a vegetative tissue, drought tolerance, nitrogenuptake, root lodging, harvest index, stalk lodging, plant height, earheight and ear length. The alteration of at least one agronomiccharacteristic may be an increase in yield, greenness or biomass.

In any of the preceding methods or any other embodiments of methods ofthe present invention, the plant may exhibit the alteration of at leastone agronomic characteristic when compared, under water limitingconditions, to a control plant not comprising said recombinant DNAconstruct (or said suppression DNA construct).

In any of the preceding methods or any other embodiments of methods ofthe present invention, alternatives exist for introducing into aregenerable plant cell a recombinant DNA construct comprising apolynucleotide operably linked to at least one regulatory sequence. Forexample, one may introduce into a regenerable plant cell a regulatorysequence (such as one or more enhancers, optionally as part of atransposable element), and then screen for an event in which theregulatory sequence is operably linked to an endogenous gene encoding apolypeptide of the instant invention.

The introduction of recombinant DNA constructs of the present inventioninto plants may be carried out by any suitable technique, including butnot limited to direct DNA uptake, chemical treatment, electroporation,microinjection, cell fusion, infection, vector-mediated DNA transfer,bombardment, or Agrobacterium-mediated transformation.

Techniques are set forth below in the Examples below for transformationof maize plant cells and soybean plant cells.

Other methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants include those published forcotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No.5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011,McCabe et. al., Bio/Technology 6:923 (1988), Christou et al., PlantPhysiol. 87:671 674 (1988)); Brassica (U.S. Pat. No. 5,463,174); peanut(Cheng et al., Plant Cell Rep. 15:653 657 (1996), McKently et al., PlantCell Rep. 14:699 703 (1995)); papaya; and pea (Grant et al., Plant CellRep. 15:254 258, (1995)).

Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have also been reported, for example,transformation and plant regeneration as achieved in asparagus (Bytebieret al., Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan andLemaux, Plant Physiol 104:37 (1994)); maize (Rhodes et al., Science240:204 (1988), Gordon-Kamm et al., Plant Cell 2:603 618 (1990), Frommet al., Bio/Technology 8:833 (1990), Koziel et al., Bio/Technology 11:194, (1993), Armstrong et al., Crop Science 35:550 557 (1995)); oat(Somers et al., Bio/Technology 10: 15 89 (1992)); orchard grass (Horn etal., Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., TheorAppl.Genet. 205:34, (1986); Part et al., Plant Mol. Biol. 32:1135 1148,(1996); Abedinia et al., Aust. J. Plant Physiol. 24:133 141 (1997);Zhang and Wu, Theor. Appl. Genet. 76:835 (1988); Zhang et al. Plant CellRep. 7:379, (1988); Battraw and Hall, Plant Sci. 86:191 202 (1992);Christou et al., Bio/Technology 9:957 (1991)); rye (De la Pena et al.,Nature 325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409(1992)); tall fescue (Wang et al., Bio/Technology 10:691 (1992)), andwheat (Vasil et al., Bio/Technology 10:667 (1992); U.S. Pat. No.5,631,152).

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, In: Methods for PlantMolecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif.,(1988)). This regeneration and growth process typically includes thesteps of selection of transformed cells, culturing those individualizedcells through the usual stages of embryonic development through therooted plantlet stage. Transgenic embryos and seeds are similarlyregenerated. The resulting transgenic rooted shoots are thereafterplanted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous isolated nucleic acid fragment that encodes a protein ofinterest is well known in the art. The regenerated plants may beself-pollinated to provide homozygous transgenic plants. Otherwise,pollen obtained from the regenerated plants is crossed to seed-grownplants of agronomically important lines. Conversely, pollen from plantsof these important lines is used to pollinate regenerated plants. Atransgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

EXAMPLES

The present invention is further illustrated in the following Examples,in which parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating embodiments of the invention, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Creation of an Arabidopsis Population with Activation-TaggedGenes

An 18.5-kb T-DNA based binary construct was created, pHSbarENDs2 (FIG.1; SEQ ID NO:31), that contains four multimerized enhancer elementsderived from the Cauliflower Mosaic Virus 35S promoter (corresponding tosequences −341 to −64, as defined by Odell et al., Nature 313:810-812(1985)). The construct also contains vector sequences (pUC9) and apolylinker to allow plasmid rescue, transposon sequences (Ds) toremobilize the T-DNA, and the bar gene to allow for glufosinateselection of transgenic plants. In principle, only the 10.8-kb segmentfrom the right border (RB) to left border (LB) inclusive will betransferred into the host plant genome. Since the enhancer elements arelocated near the RB, they can induce cis-activation of genomic locifollowing T-DNA integration.

Arabidopsis activation-tagged populations were created by whole plantAgrobacterium transformation. The pHSbarENDs2 construct was transformedinto Agrobacterium tumefaciens strain C58, grown in LB at 25° C. toOD600˜1.0. Cells were then pelleted by centrifugation and resuspended inan equal volume of 5% sucrose/0.05% Silwet L-77 (OSI Specialties, Inc).At early bolting, soil grown Arabidopsis thaliana ecotype Col-0 were topwatered with the Agrobacterium suspension. A week later, the same plantswere top watered again with the same Agrobacterium strain insucrose/Silwet. The plants were then allowed to set seed as normal. Theresulting T1 seed were sown on soil, and transgenic seedlings wereselected by spraying with glufosinate (Finale®; AgrEvo; BayerEnvironmental Science). A total of 100,000 glufosinate resistant T1seedlings were selected. T2 seed from each line was kept separate.

Example 2 Screens to Identify Lines with Enhanced Drought Tolerance

Quantitative Drought Screen:

From each of 96,000 separate T1 activation-tagged lines, nineglufosinate resistant T2 plants are sown, each in a single pot onScotts® Metro-Mix® 200 soil. Flats are configured with 8 square potseach. Each of the square pots is filled to the top with soil. Each pot(or cell) is sown to produce 9 glufosinate resistant seedlings in a 3×3array.

The soil is watered to saturation and then plants are grown understandard conditions (i.e., 16 hour light, 8 hour dark cycle; 22° C.;˜60% relative humidity). No additional water is given.

Digital images of the plants are taken at the onset of visible droughtstress symptoms. Images are taken once a day (at the same time of day),until the plants appear dessicated. Typically, four consecutive days ofdata is captured.

Color analysis is employed for identifying potential drought tolerantlines. Color analysis can be used to measure the increase in thepercentage of leaf area that falls into a yellow color bin. Using hue,saturation and intensity data (“HSI”), the yellow color bin consists ofhues 35 to 45.

Maintenance of leaf area is also used as another criterion foridentifying potential drought tolerant lines, since Arabidopsis leaveswilt during drought stress. Maintenance of leaf area can be measured asreduction of rosette leaf area over time.

Leaf area is measured in terms of the number of green pixels obtainedusing the LemnaTec imaging system. Activation-tagged and control (e.g.,wild-type) plants are grown side by side in flats that contain 72 plants(9 plants/pot). When wilting begins, images are measured for a number ofdays to monitor the wilting process. From these data wilting profilesare determined based on the green pixel counts obtained over fourconsecutive days for activation-tagged and accompanying control plants.The profile is selected from a series of measurements over the four dayperiod that gives the largest degree of wilting. The ability towithstand drought is measured by the tendency of activation-taggedplants to resist wilting compared to control plants.

LemnaTec HTSBonitUV software is used to analyze CCD images. Estimates ofthe leaf area of the Arabidopsis plants are obtained in terms of thenumber of green pixels. The data for each image is averaged to obtainestimates of mean and standard deviation for the green pixel counts foractivation-tagged and wild-type plants. Parameters for a noise functionare obtained by straight line regression of the squared deviation versusthe mean pixel count using data for all images in a batch. Errorestimates for the mean pixel count data are calculated using the fitparameters for the noise function. The mean pixel counts foractivation-tagged and wild-type plants are summed to obtain anassessment of the overall leaf area for each image. The four-dayinterval with maximal wilting is obtained by selecting the interval thatcorresponds to the maximum difference in plant growth. The individualwilting responses of the activation-tagged and wild-type plants areobtained by normalization of the data using the value of the green pixelcount of the first day in the interval. The drought tolerance of theactivation-tagged plant compared to the wild-type plant is scored bysumming the weighted difference between the wilting response ofactivation-tagged plants and wild-type plants over day two to day four;the weights are estimated by propagating the error in the data. Apositive drought tolerance score corresponds to an activation-taggedplant with slower wilting compared to the wild-type plant. Significanceof the difference in wilting response between activation-tagged andwild-type plants is obtained from the weighted sum of the squareddeviations.

Lines with a significant delay in yellow color accumulation and/or withsignificant maintenance of rosette leaf area, when compared to theaverage of the whole flat, are designated as Phase 1 hits. Phase 1 hitsare re-screened in duplicate under the same assay conditions. Wheneither or both of the Phase 2 replicates show a significant difference(score of greater than 0.9) from the whole flat mean, the line is thenconsidered a validated drought tolerant line.

Example 3 Identification of Activation-Tagged Genes

Genes flanking the T-DNA insert in drought tolerant lines are identifiedusing one, or both, of the following two standard procedures: (1)thermal asymmetric interlaced (TAIL) PCR (Liu et al., (1995), Plant J.8:457-63); and (2) SAIFF PCR (Siebert et al., (1995) Nucleic Acids Res.23:1087-1088). In lines with complex multimerized T-DNA inserts, TAILPCR and SAIFF PCR may both prove insufficient to identify candidategenes. In these cases, other procedures, including inverse PCR, plasmidrescue and/or genomic library construction, can be employed.

A successful result is one where a single TAIL or SAIFF PCR fragmentcontains a T-DNA border sequence and Arabidopsis genomic sequence.

Once a tag of genomic sequence flanking a T-DNA insert is obtained,candidate genes are identified by alignment to publicly availableArabidopsis genome sequence.

Specifically, the annotated gene nearest the 35S enhancer elements/T-DNARB are candidates for genes that are activated.

To verify that an identified gene is truly near a T-DNA and to rule outthe possibility that the TAIL/SAIFF fragment is a chimeric cloningartifact, a diagnostic PCR on genomic DNA is done with one oligo in theT-DNA and one oligo specific for the candidate gene. Genomic DNA samplesthat give a PCR product are interpreted as representing a T-DNAinsertion. This analysis also verifies a situation in which more thanone insertion event occurs in the same line, e.g., if multiple differinggenomic fragments are identified in TAIL and/or SAIFF PCR analyses.

Example 4A Identification of Activation-Tagged Ferrochelatase-I (FeC-I)Gene

An activation-tagged line (No. 105998) showing drought tolerance wasfurther analyzed. DNA from the line was extracted, and genes flankingthe T-DNA insert in the mutant line were identified using SAIFF PCR(Siebert et al., Nucleic Acids Res. 23:1087-1088 (1995)). A PCRamplified fragment was identified that contained T-DNA border sequenceand Arabidopsis genomic sequence. Genomic sequences flanking the T-DNAinsert was obtained, and the candidate gene was identified by alignmentto the completed Arabidopsis genome. For a given T-DNA integrationevent, the annotated gene nearest the 35S enhancer elements/T-DNA RB wasthe candidate for gene that is activated in the line. In the case ofline 105998, the gene nearest the 35S enhancers at the integration sitewas At5g26030 (SEQ ID NO:1; NCBI GI No. 511080), encoding aferrochelatase-I protein (SEQ ID NO:2; NCBI GI No. 511081).

Example 4B Assay for Expression Level of Candidate Drought ToleranceGenes

A functional activation-tagged allele should result in eitherup-regulation of the candidate gene in tissues where it is normallyexpressed, ectopic expression in tissues that do not normally expressthat gene, or both.

Expression levels of the candidate genes in the cognate mutant line vs.wild-type are compared. A standard RT-PCR procedure, such as theQuantiTect® Reverse Transcription Kit from Qiagen®, is used. RT-PCR ofthe actin gene is used as a control to show that the amplification andloading of samples from the mutant line and wild-type are similar.

Assay conditions are optimized for each gene. Expression levels arechecked in mature rosette leaves. If the activation-tagged alleleresults in ectopic expression in other tissues (e.g., roots), it is notdetected by this assay. As such, a positive result is useful but anegative result does not eliminate a gene from further analysis.

Example 5A Validation of Arabidopsis Candidate Gene At5g26030(Ferrochelatase-I) Via Transformation into Arabidopsis

Candidate genes can be transformed into Arabidopsis and overexpressedunder the 35S promoter. If the same or similar phenotype is observed inthe transgenic line as in the parent activation-tagged line, then thecandidate gene is considered to be a validated “lead gene” inArabidopsis.

The candidate Arabidopsis ferrochelatase-I gene (At5g26030; SEQ ID NO:1;NCBI GI No. 511080) was tested for its ability to confer droughttolerance in the following manner.

A 16.8-kb T-DNA based binary vector, called pBC-yellow (SEQ ID NO:34;FIG. 4), was constructed with a 1.3-kb 35S promoter immediately upstreamof the INVITROGEN™ GATEWAY® C1 conversion insert. The vector alsocontains the RD29a promoter driving expression of the gene for ZS-Yellow(INVITROGEN™), which confers yellow fluorescence to transformed seed.

The At5g26030 cDNA protein-coding region was amplified by RT-PCR withthe following primers:

(1) At5g26030-5′attB forward primer (SEQ ID NO: 42):TTAAACAAGTTTGTACAAAAAAGCAGGCTCAACAATGCAGGCAACGG CTTTATCA(2) At5g26030-3′attB reverse primer (SEQ ID NO: 43):TTAAACCACTTTGTACAAGAAAGCTGGGTCTATAGGTTCCGGAACGC ATG

The forward primer contains the attB1 sequence(ACAAGTTTGTACAAAAAAGCAGGCT; SEQ ID NO:40) and a consensus Kozak sequence(CAACA) adjacent to the first 21 nucleotides of the protein-codingregion, beginning with the ATG start codon, of said cDNA.

The reverse primer contains the attB2 sequence(ACCACTTTGTACAAGAAAGCTGGGT; SEQ ID NO:41) adjacent to the reversecomplement of the last 21 nucleotides of the protein-coding region,beginning with the reverse complement of the stop codon, of said cDNA.

Using the INVITROGEN™ GATEWAY® CLONASE™ technology, a BP RecombinationReaction was performed with pDONR™/Zeo (SEQ ID NO:32; FIG. 2). Thisprocess removed the bacteria lethal ccdB gene, as well as thechloramphenicol resistance gene (CAM) from pDONR™/Zeo and directionallycloned the PCR product with flanking attB1 and attB2 sites creating anentry clone, PHP31052. This entry clone was used for a subsequent LRRecombination Reaction with a destination vector, as follows.

A 16.8-kb T-DNA based binary vector (destination vector), calledpBC-yellow (SEQ ID NO:34; FIG. 4), was constructed with a 1.3-kb 35Spromoter immediately upstream of the INVITROGEN™ GATEWAY® C1 conversioninsert, which contains the bacterial lethal ccdB gene as well as thechloramphenicol resistance gene (CAM) flanked by attR1 and attR2sequences. The vector also contains the RD29a promoter drivingexpression of the gene for ZS-Yellow (INVITROGEN™), which confers yellowfluorescence to transformed seed. Using the INVITROGEN™ GATEWAY®technology, an LR Recombination Reaction was performed on the PHP31052entry clone, containing the directionally cloned PCR product, andpBC-yellow. This allowed for rapid and directional cloning of thecandidate gene behind the 35S promoter in pBC-yellow to create the 35Spromoter::At5g26030 expression construct, pBC-Yellow-At5g26030.

Applicants then introduced the 35S promoter::At5g26030 expressionconstruct into wild-type Arabidopsis ecotype Col-0, using the sameAgrobacterium-mediated transformation procedure described in Example 1.Transgenic T1 seeds were selected by yellow fluorescence, and T1 seedswere plated next to wild-type seeds and grown under water limitingconditions. Growth conditions and imaging analysis were as described inExample 2. It was found that the original drought tolerance phenotypefrom activation tagging could be recapitulated in wild-type Arabidopsisplants that were transformed with a construct where At5g26030 wasdirectly expressed by the 35S promoter. The drought tolerance score, asdetermined by the method of Example 2, was 5.3.

Example 5B Validation of Arabidopsis Candidate Gene At2g30390(Ferrochelatase-II) Via Transformation into Arabidopsis

A cDNA encoding ferrochelatase-I from Arabidopsis was previously clonedby functional complementation of a yeast mutant (Smith et al. 1994 JBiol Chem 269:13405-13413). Subsequently, a second ferrochelataseisoform, ferrochelatase-II, was found in Arabidopsis (Chow et al. 1998Plant J 15:531-541). Two forms of ferrochelatase also have beenidentified in cucumber, Cucumis sativus (Miyamoto et al. 1994 PlantPhysiol 105:769-770; and Suzuki et al. 2002 J Biol Chem 277:4731-4737).The C-terminal region of ferrochelatase-II, but not ferrochelatase-I,contains a conserved motif found in light-harvesting chlorophyllproteins (Suzuki et al. 2002 J Biol Chem 277:4731-4737). The C-terminalregion of the ferrochelatase from the cyanobacteria Synechocystis (NCBIGI NO. 1708186) has sequence homology to this conserved motif.

The Arabidopsis ferrochelatase-II gene (At2g30390) was selected as asecond candidate gene, and was tested for its ability to confer droughttolerance in the following manner.

A 16.8-kb T-DNA based binary vector, called pBC-yellow (SEQ ID NO:34;FIG. 4), was constructed with a 1.3-kb 35S promoter immediately upstreamof the INVITROGEN™ GATEWAY® C1 conversion insert. The vector alsocontains the RD29a promoter driving expression of the gene for ZS-Yellow(INVITROGEN™), which confers yellow fluorescence to transformed seed.

The At2g30390 cDNA protein-coding region was amplified by RT-PCR withthe following primers:

(3) At2g30390-5′attB forward primer (SEQ ID NO: 44):TTAAACAAGTTTGTACAAAAAAGCAGGCTCAACAATGAATTGCCCAG CCATGACT(4) At2g30390-3′attB reverse primer (SEQ ID NO: 45):TTAAACCACTTTGTACAAGAAAGCTGGGTTTATAATGAAGGCAAGAT GCC

The forward primer contains the attB1 sequence(ACAAGTTTGTACAAAAAAGCAGGCT; SEQ ID NO:40) and a consensus Kozak sequence(CAACA) adjacent to the first 21 nucleotides of the protein-codingregion, beginning with the ATG start codon, of said cDNA.

The reverse primer contains the attB2 sequence(ACCACTTTGTACAAGAAAGCTGGGT; SEQ ID NO:41) adjacent to the reversecomplement of the last 21 nucleotides of the protein-coding region,beginning with the reverse complement of the stop codon, of said cDNA.

Using the INVITROGEN™ GATEWAY® CLONASE™ technology, a BP RecombinationReaction was performed with pDONR™/Zeo (SEQ ID NO:32; FIG. 2). Thisprocess removed the bacteria lethal ccdB gene, as well as thechloramphenicol resistance gene (CAM) from pDONR™/Zeo and directionallycloned the PCR product with flanking attB1 and attB2 sites creating anentry clone, PHP-Entry-At2g30390. This entry clone was used for asubsequent LR Recombination Reaction with a destination vector, asfollows.

A 16.8-kb T-DNA based binary vector (destination vector), calledpBC-yellow (SEQ ID NO:34; FIG. 4), was constructed with a 1.3-kb 35Spromoter immediately upstream of the INVITROGEN™ GATEWAY® C1 conversioninsert, which contains the bacterial lethal ccdB gene as well as thechloramphenicol resistance gene (CAM) flanked by attR1 and attR2sequences. The vector also contains the RD29a promoter drivingexpression of the gene for ZS-Yellow (INVITROGEN™), which confers yellowfluorescence to transformed seed. Using the INVITROGEN™ GATEWAY®technology, an LR Recombination Reaction was performed on thePHP-Entry-At2g30390 entry clone, containing the directionally cloned PCRproduct, and pBC-yellow. This allowed for rapid and directional cloningof the candidate gene behind the 35S promoter in pBC-yellow to createthe 35S promoter::At2g30390 expression construct, pBC-Yellow-At2g30390.

Applicants then introduced the 35S promoter::At2g30390 expressionconstruct into wild-type Arabidopsis ecotype Col-0, using the sameAgrobacterium-mediated transformation procedure described in Example 1.Transgenic T1 seeds were selected by yellow fluorescence, and T1 seedswere plated next to wild-type seeds and grown under water limitingconditions. Growth conditions and imaging analysis were as described inExample 2. It was found that the original drought tolerance phenotypefrom activation tagging could be recapitulated in wild-type Arabidopsisplants that were transformed with a construct where At2g30390 wasdirectly expressed by the 35S promoter. The drought tolerance score, asdetermined by the method of Example 2, was 3.7.

Example 6A Preparation of cDNA Libraries and Isolation and Sequencing ofcDNA Clones

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in UNI-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The UNI-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript®. In addition, thecDNAs may be introduced directly into precut Bluescript® II SK(+)vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followedby transfection into DH10B cells according to the manufacturer'sprotocol (GIBCO BRL Products). Once the cDNA inserts are in plasmidvectors, plasmid DNAs are prepared from randomly picked bacterialcolonies containing recombinant pBluescript® plasmids, or the insertcDNA sequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

Confirmed templates are transposed via the Primer Island transpositionkit (PE Applied Biosystems, Foster City, Calif.) which is based upon theSaccharomyces cerevisiae Ty1 transposable element (Devine and Boeke(1994) Nucleic Acids Res. 22:3765-3772). The in vitro transpositionsystem places unique binding sites randomly throughout a population oflarge DNA molecules. The transposed DNA is then used to transform DH10Belectro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.)via electroporation. The transposable element contains an additionalselectable marker (named DHFR; Fling and Richards (1983) Nucleic AcidsRes. 11:5147-5158), allowing for dual selection on agar plates of onlythose subclones containing the integrated transposon. Multiple subclonesare randomly selected from each transposition reaction, plasmid DNAs areprepared via alkaline lysis, and templates are sequenced (ABI Prism®dye-terminator ReadyReaction mix) outward from the transposition eventsite, utilizing unique primers specific to the binding sites within thetransposon.

Sequence data is collected (ABI Prism® Collections) and assembled usingPhred and Phrap (Ewing et al. (1998) Genome Res. 8:175-185; Ewing andGreen (1998) Genome Res. 8:186-194). Phred is a public domain softwareprogram which re-reads the ABI sequence data, re-calls the bases,assigns quality values, and writes the base calls and quality valuesinto editable output files. The Phrap sequence assembly program usesthese quality values to increase the accuracy of the assembled sequencecontigs. Assemblies are viewed by the Consed sequence editor (Gordon etal. (1998) Genome Res. 8:195-202).

In some of the clones the cDNA fragment may correspond to a portion ofthe 3′-terminus of the gene and does not cover the entire open readingframe. In order to obtain the upstream information one of two differentprotocols is used. The first of these methods results in the productionof a fragment of DNA containing a portion of the desired gene sequencewhile the second method results in the production of a fragmentcontaining the entire open reading frame. Both of these methods use tworounds of PCR amplification to obtain fragments from one or morelibraries. The libraries some times are chosen based on previousknowledge that the specific gene should be found in a certain tissue andsome times are randomly-chosen. Reactions to obtain the same gene may beperformed on several libraries in parallel or on a pool of libraries.Library pools are normally prepared using from 3 to 5 differentlibraries and normalized to a uniform dilution. In the first round ofamplification both methods use a vector-specific (forward) primercorresponding to a portion of the vector located at the 5′-terminus ofthe clone coupled with a gene-specific (reverse) primer. The firstmethod uses a sequence that is complementary to a portion of the alreadyknown gene sequence while the second method uses a gene-specific primercomplementary to a portion of the 3′-untranslated region (also referredto as UTR). In the second round of amplification a nested set of primersis used for both methods. The resulting DNA fragment is ligated into apBluescript® vector using a commercial kit and following themanufacturer's protocol. This kit is selected from many available fromseveral vendors including INVITROGEN™ (Carlsbad, Calif.), PromegaBiotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmidDNA is isolated by alkaline lysis method and submitted for sequencingand assembly using Phred/Phrap, as above.

Example 7 Identification of cDNA Clones

cDNA clones encoding ferrochelatases can be identified by conductingBLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.Biol. 215:403-410; see also the explanation of the BLAST algorithm onthe world wide web site for the National Center for BiotechnologyInformation at the National Library of Medicine of the NationalInstitutes of Health) searches for similarity to amino acid sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). The DNAsequences from clones can be translated in all reading frames andcompared for similarity to all publicly available protein sequencescontained in the “nr” database using the BLASTX algorithm (Gish andStates (1993) Nat. Genet. 3:266-272) provided by the NCBI. Thepolypeptides encoded by the cDNA sequences can be analyzed forsimilarity to all publicly available amino acid sequences contained inthe “nr” database using the BLASTP algorithm provided by the NationalCenter for Biotechnology Information (NCBI). For convenience, theP-value (probability) or the E-value (expectation) of observing a matchof a cDNA-encoded sequence to a sequence contained in the searcheddatabases merely by chance as calculated by BLAST are reported herein as“pLog” values, which represent the negative of the logarithm of thereported P-value or E-value. Accordingly, the greater the pLog value,the greater the likelihood that the cDNA-encoded sequence and the BLAST“hit” represent homologous proteins.

ESTs sequences can be compared to the Genbank database as describedabove. ESTs that contain sequences more 5- or 3-prime can be found byusing the BLASTn algorithm (Altschul et al (1997) Nucleic Acids Res.25:3389-3402.) against the Du Pont proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5 or 3 prime direction. Once the most 5-prime ESTis identified, its complete sequence can be determined by Full InsertSequencing as described above. Homologous genes belonging to differentspecies can be found by comparing the amino acid sequence of a knowngene (from either a proprietary source or a public database) against anEST database using the tBLASTn algorithm. The tBLASTn algorithm searchesan amino acid query against a nucleotide database that is translated inall 6 reading frames. This search allows for differences in nucleotidecodon usage between different species, and for codon degeneracy.

Example 8 Characterization of cDNA Clones Encoding Ferrochetalases

cDNA libraries representing mRNAs from various tissues of Sugar Beet,Canola, Maize, Rice, Soybean, Wheat and Catmint were prepared and cDNAclones encoding ferrochelatases were identified. The characteristics ofthe libraries are described below.

TABLE 3 cDNA Libraries from Sugar Beet, Canola, Maize, Rice, Soybean,Wheat and Catmint Library Description Clone ebs1c Sugar Beet, shoot andphloem specific ebs1c.pk002.n16:fis genes ebb1c Immature buds of Canola,Rf gene knock ebb1c.pk006.j11:fis out mutant line, 02SM2 cfp3n MaizeEar, pooled V10-V14-v16-VT, cfp3n.pk004.f12:fis Full-length enrichednormalized cfp5n Maize Kernel, pooled stages, cfp5n.pk009.j16:fisFull-length enriched, normalized rl0n Rice 15 Day Old Leaf*rl0n.pk117.h21:fis se3 Soybean Embryo, 17 Days After se3.pk0034.e10:fisFlowering etb1n Normalized tulip bulb library etb1n.pk002.n16:fis wlp1cWheat (Triticum aestivum, Hi Line) wlp1c.pk002.p10:fis lemma and paleaecl1c Catmint (Nepeta racemosa) cDNA library ecl1c.pk005.l15:fis fromyoung leaf tissue cfp6n Maize Leaf and Seed pooled, Full-lengthcfp6n.pk072.n9:fis enriched normalized wlp1c Wheat (Triticum aestivum,Hi Line) wlp1c.pk004.d13:fis lemma and palea wpa1c Wheat (Triticumaestivum) pre-meiotic wpa1c.pk014.g4:fis anthers *These libraries werenormalized essentially as described in U.S. Pat. No. 5,482,845

Two of the cDNA clones listed in Table 3 had anomalies. The cloneebb1c.pk006.j11:fis contains an unspliced intron at nucleotides 386-481of SEQ ID NO:5. The polypeptide encoded by the two exons surrounding theintron is given in SEQ ID NO:6. Additionally, the cloneecl1c.pk005.l15:fis has a single base deletion at nucleotide 442, whichresults in a frame-shift in the corresponding translation. Based oncomparison to a highly similar clone cDNA clone from grape,veb1c.pk008.p6 (411 base pairs), a “T” nucleotide was added to theecl1c.pk005.l15:fis sequence at position 442. The modified nucleotidesequence is presented in SEQ ID NO:23, which encodes the amino acidsequence of SEQ ID NO:24.

The BLAST search using the sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to theferrochelatases from various organisms. As shown in Table 4 and FIGS.10A-10C, certain cDNAs encoded polypeptides similar to ferrochelatase-Iproteins from Arabidopsis (GI No. 511081; SEQ ID NO:2; FeC-I), barley(GI No. 2460251; SEQ ID NO:48; FeC-I), rice (GI No. 113631036; SEQ IDNO:49; FeC-I), and grape (GI No. 147818793; SEQ ID NO:50; FeC-I). Asshown in Table 6 and FIGS. 12A-12C, certain cDNAs encoded polypeptidessimilar to ferrochelatase-II proteins from Arabidopsis (GI No. 15227742;SEQ ID NO:20; FeC-II), rice (GI No. 115463419; SEQ ID NO:51; FeC-II),cucumber (GI No. 12082085; SEQ ID NO:52; FeC-II), tobacco (GI No.15147828; SEQ ID NO:53; FeC-II) and a ferrochelatse-II-like protein fromSynechocystis (GI No. 1708186; SEQ ID NO:54; FeC-II-like).

Shown in Tables 4 and 6 (non-patent literature) and Tables 5 and 7(patent literature) are the BLASTP results for the amino acid sequencesderived from the nucleotide sequences of the entire cDNA inserts(“Full-Insert Sequence” or “FIS”) of the clones listed in Table 3. EachcDNA insert encodes an entire or functional protein (“Complete GeneSequence” or “CGS”). Also shown in Tables 4-7 are the percent sequenceidentity values for each pair of amino acid sequences:

TABLE 4 BLASTP Results for Ferrochelatase-I Polypeptides Percent BLASTPSe- Sequence NCBI GI No. pLog of quence (SEQ ID NO) Status (SEQ ID NO)E-value Identity ebs1c.pk002.n16 (FIS) CGS 147818793 >180 72.6 (SEQ IDNO: 4) (SEQ ID NO: 50) ebb1c.pk006.j11 (FIS) CGS 511081 >180 83.3 (SEQID NO: 6) (SEQ ID NO: 2) cfp3n.pk004.f12 (FIS) CGS 113631036 >180 84.0(SEQ ID NO: 8) (SEQ ID NO: 49) cfp5n.pk009.j16 (FIS) CGS 113631036 >18081.7 (SEQ ID NO: 10) (SEQ ID NO: 49) rl0n.pk117.h21 (FIS) CGS113631036 >180 100 (SEQ ID NO: 12) (SEQ ID NO: 49) se3.pk0034.e10 (FIS)CGS 147818793 >180 74.2 (SEQ ID NO: 14) (SEQ ID NO: 50) etb1n.pk002.n16(FIS) CGS 113631036 180 69.5 (SEQ ID NO: 16) (SEQ ID NO: 49)wlp1c.pk002.p10 (FIS) CGS 2460251 >180 98.3 (SEQ ID NO: 18) (SEQ ID NO:48)

TABLE 5 BLASTP Results for Ferrochelatase-I Polypeptides PercentSequence Reference BLASTP Sequence (SEQ ID NO) Status (SEQ ID NO) pLogof E-value Identity ebs1c.pk002.n16 (FIS) CGS SEQ ID NO: 240025 >18068.8 (SEQ ID NO: 4) of US2004031072 (SEQ ID NO: 57) ebb1c.pk006.j11(FIS) CGS SEQ ID NO: 20 of >180 83.3 (SEQ ID NO: 6) JP2001190168-A (SEQID NO: 58) cfp3n.pk004.f12 (FIS) CGS SEQ ID NO: 13029 of >180 99.4 (SEQID NO: 8) US2006150283 (SEQ ID NO: 59) cfp5n.pk009.j16 (FIS) CGS SEQ IDNO: 7745 of >180 100 (SEQ ID NO: 10) US2006150283 (SEQ ID NO: 60)rl0n.pk117.h21 (FIS) CGS SEQ ID NO: 46156 >180 100 (SEQ ID NO: 12) ofJP2005185101 (SEQ ID NO: 61) se3.pk0034.e10 (FIS) CGS SEQ ID NO:240025 >180 100 (SEQ ID NO: 14) of US2004031072 (SEQ ID NO: 57)etb1n.pk002.n16 (FIS) CGS SEQ ID NO: 46156 >180 69.5 (SEQ ID NO: 16) ofJP2005185101 (SEQ ID NO: 61) wlp1c.pk002.p10 (FIS) CGS SEQ ID NO:46156 >180 84.6 (SEQ ID NO: 18) of JP2005185101 (SEQ ID NO: 61)

TABLE 6 BLASTP Results for Ferrochelatase-II Polypeptides Percent BLASTPSe- Sequence NCBI GI No. pLog of quence (SEQ ID NO) Status (SEQ ID NO)E-value Identity ecl1c.pk005.l15 (FIS) CGS 12082085 >180 78.6 (SEQ IDNO: 24) (SEQ ID NO: 52) cfp6n.pk072.n9 (FIS) CGS 115463419 >180 90.5(SEQ ID NO: 26) (SEQ ID NO: 51) wlp1c.pk004.d13 (FIS) CGS 115463419 >18082.9 (SEQ ID NO: 28) (SEQ ID NO: 51) wpa1c.pk014.g4 (FIS) CGS115463419 >180 83.3 (SEQ ID NO: 30) (SEQ ID NO: 51)

TABLE 7 BLASTP Results for Ferrochelatase-II Polypeptides BLASTP PercentSequence Reference pLog of Sequence (SEQ ID NO) Status (SEQ ID NO)E-value Identity ecl1c.pk005.l15 CGS SEQ ID NO: >180 77.4 (FIS) (SEQ IDNO: 52154 of 24) JP2005185101 (SEQ ID NO: 62) cfp6n.pk072.n9 CGS SEQ IDNO: >180 99.6 (FIS) (SEQ ID NO: 72746 of 26) US2004034888 (SEQ ID NO:63) wlp1c.pk004.d13 CGS SEQ ID NO: >180 82.9 (FIS) (SEQ ID NO: 52154 of28) JP2005185101 (SEQ ID NO: 62) wpa1c.pk014.g4 CGS SEQ ID NO: >180 83.3(FIS) (SEQ ID NO: 52154 of 30) JP2005185101 (SEQ ID NO: 62)

FIGS. 10A-10C present an alignment of the amino acid sequences offerrochelatase-I proteins set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12,14, 16, 18, 48, 49 and 50. FIG. 11 presents the percent sequenceidentities and divergence values for each sequence pair presented inFIGS. 10A and 10B.

FIGS. 12A-12C present an alignment of the amino acid sequences offerrochelatase-II proteins set forth in SEQ ID NOs:20, 22, 24, 26, 28,30, 51, 52, 53 and 54. FIG. 13 presents the percent sequence identitiesand divergence values for each sequence pair presented in FIGS. 12A and12B.

Sequence alignments and percent identity calculations were performedusing the Megalign® program of the LASERGENE® bioinformatics computingsuite (DNASTAR® Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal V method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

Import experiments into isolated chloroplasts and mitochondria showedthat the Arabidopsis ferrochelatase-II gene encodes a precursor which isimported solely into the chloroplast, in contrast to Arabidopsisferrochelatase-I which is targeted to both organelles (Chow et al. 1997J Biol Chem 272:27565-27571; Chow et al. 1998 Plant J 15:531-541). Theferrochelatase from Synechocystis (GI No. 1708186; SEQ ID NO:54) is moresimilar to the ferrechelatase-II isoform.

Sequence alignments and BLAST scores and probabilities indicate that thenucleic acid fragments comprising the instant cDNA clones encodeferrochelatases.

Example 9 Preparation of a Plant Expression Vector Containing a Homologto the Arabidopsis Lead Gene

Sequences homologous to the Arabidopsis ferrochelatase-I polypeptide canbe identified using sequence comparison algorithms such as BLAST (BasicLocal Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410(1993); see also the explanation of the BLAST algorithm on the worldwide web site for the National Center for Biotechnology Information atthe National Library of Medicine of the National Institutes of Health).Sequences encoding homologous ferrochelatases can be PCR-amplified byeither of the following methods.

Method 1 (RNA-based): If the 5′ and 3′ sequence information for theprotein-coding region of a gene encoding a ferrochelatase homolog isavailable, gene-specific primers can be designed as outlined in Example5. RT-PCR can be used with plant RNA to obtain a nucleic acid fragmentcontaining the protein-coding region flanked by attB1 (SEQ ID NO:40) andattB2 (SEQ ID NO:41) sequences. The primer may contain a consensus Kozaksequence (CAACA) upstream of the start codon.

Method 2 (DNA-based): Alternatively, if a cDNA clone is available for agene encoding a ferrochelatase homolog, the entire cDNA insert(containing 5′ and 3′ non-coding regions) can be PCR amplified. Forwardand reverse primers can be designed that contain either the attB1sequence and vector-specific sequence that precedes the cDNA insert orthe attB2 sequence and vector-specific sequence that follows the cDNAinsert, respectively. For a cDNA insert cloned into the vectorpBulescript SK+, the forward primer VC062 (SEQ ID NO:46) and the reverseprimer VC063 (SEQ ID NO:47) can be used.

Methods 1 and 2 can be modified according to procedures known by oneskilled in the art. For example, the primers of Method 1 may containrestriction sites instead of attB1 and attB2 sites, for subsequentcloning of the PCR product into a vector containing attB1 and attB2sites. Additionally, Method 2 can involve amplification from a cDNAclone, a lambda clone, a BAC clone or genomic DNA.

A PCR product obtained by either method above can be combined with theGATEWAY® donor vector, such as pDONR™/Zeo (INVITROGEN™; FIG. 2; SEQ IDNO:32) or pDONR™221 (INVITROGEN™; FIG. 3; SEQ ID NO:33), using a BPRecombination Reaction. This process removes the bacteria lethal ccdBgene, as well as the chloramphenicol resistance gene (CAM) frompDONR™221 and directionally clones the PCR product with flanking attB1and attB2 sites to create an entry clone. Using the INVITROGEN™ GATEWAY®CLONASE™ technology, the sequence encoding the homologous ferrochelatasefrom the entry clone can then be transferred to a suitable destinationvector, such as pBC-Yellow (FIG. 4; SEQ ID NO:34), PHP27840 (FIG. 5; SEQID NO:35) or PHP23236 (FIG. 6; SEQ ID NO:36), to obtain a plantexpression vector for use with Arabidopsis, soybean and corn,respectively.

The attP1 and attP2 sites of donor vectors pDONR™/Zeo or pDONR™221 areshown in FIGS. 2 and 3, respectively. The attR1 and attR2 sites ofdestination vectors pBC-Yellow, PHP27840 and PHP23236 are shown in FIGS.4, 5 and 6, respectively.

Alternatively a MultiSite GATEWAY® LR recombination reaction betweenmultiple entry clones and a suitable destination vector can be performedto create an expression vector.

Example 10 Preparation of Soybean Expression Vectors and Transformationof Soybean with Validated Arabidopsis Lead Genes

Soybean plants can be transformed to overexpress a validated Arabidopsislead gene or the corresponding homologs from various species in order toexamine the resulting phenotype.

The same GATEWAY® entry clone described in Example 5 can be used todirectionally clone each gene into the PHP27840 vector (SEQ ID NO:35;FIG. 5) such that expression of the gene is under control of the SCP1promoter.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides.

To induce somatic embryos, cotyledons, 3-5 mm in length dissected fromsurface sterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos, which produce secondary embryos, arethen excised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiply as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DUPONT™ BIOLISTIC™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromcauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. Another selectable marker gene which can be used tofacilitate soybean transformation is an herbicide-resistant acetolactatesynthase (ALS) gene from soybean or Arabidopsis. ALS is the first commonenzyme in the biosynthesis of the branched-chain amino acids valine,leucine and isoleucine. Mutations in ALS have been identified thatconvey resistance to some or all of three classes of inhibitors of ALS(U.S. Pat. No. 5,013,659; the entire contents of which are hereinincorporated by reference). Expression of the herbicide-resistant ALSgene can be under the control of a SAM synthetase promoter (U.S. PatentApplication No. US-2003-0226166-A1; the entire contents of which areherein incorporated by reference).

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

T1 plants can be subjected to a soil-based drought stress. Using imageanalysis, plant area, volume, growth rate and color analysis can betaken at multiple times before and during drought stress. Overexpressionconstructs that result in a significant delay in wilting or leaf areareduction, yellow color accumulation and/or increased growth rate duringdrought stress will be considered evidence that the Arabidopsis genefunctions in soybean to enhance drought tolerance.

Soybean plants transformed with validated genes can then be assayedunder more vigorous field-based studies to study yield enhancementand/or stability under well-watered and water-limiting conditions.

Example 11 Transformation of Maize with Validated Arabidopsis Lead GenesUsing Particle Bombardment

Maize plants can be transformed to overexpress a validated Arabidopsislead gene or the corresponding homologs from various species in order toexamine the resulting phenotype.

The same GATEWAY® entry clone described in Example 5 can be used todirectionally clone each gene into a maize transformation vector.Expression of the gene in the maize transformation vector can be undercontrol of a constitutive promoter such as the maize ubiquitin promoter(Christensen et al., (1989) Plant Mol. Biol. 12:619-632 and Christensenet al., (1992) Plant Mol. Biol. 18:675-689)

The recombinant DNA construct described above can then be introducedinto corn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a KAPTON™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a DUPONT™ BIOLISTIC™ PDS-1000/He(Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovers a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains bialaphos (5 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containingbialaphos. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing thebialaphos-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).Transgenic T0 plants can be regenerated and their phenotype determinedfollowing high throughput (“HTP”) procedures. T1 seed can be collected.

T1 plants can be subjected to a soil-based drought stress. Using imageanalysis, plant area, volume, growth rate and color analysis can betaken at multiple times before and during drought stress. Overexpressionconstructs that result in a significant delay in wilting or leaf areareduction, yellow color accumulation and/or increased growth rate duringdrought stress will be considered evidence that the Arabidopsis genefunctions in maize to enhance drought tolerance.

Example 12 Electroporation of Agrobacterium tumefaciens LBA4404

Electroporation competent cells (40 4), such as Agrobacteriumtumefaciens LBA4404 containing PHP10523 (FIG. 7; SEQ ID NO:37), arethawed on ice (20-30 min). PHP10523 contains VIR genes for T-DNAtransfer, an Agrobacterium low copy number plasmid origin ofreplication, a tetracycline resistance gene, and a Cos site for in vivoDNA bimolecular recombination. Meanwhile the electroporation cuvette ischilled on ice. The electroporator settings are adjusted to 2.1 kV. ADNA aliquot (0.5 μL parental DNA at a concentration of 0.2 μg-1.0 μg inlow salt buffer or twice distilled H₂O) is mixed with the thawedAgrobacterium tumefaciens LBA4404 cells while still on ice. The mixtureis transferred to the bottom of electroporation cuvette and kept at reston ice for 1-2 min. The cells are electroporated (Eppendorfelectroporator 2510) by pushing the “pulse” button twice (ideallyachieving a 4.0 millisecond pulse). Subsequently, 0.5 mL of roomtemperature 2×YT medium (or SOC medium) are added to the cuvette andtransferred to a 15 mL snap-cap tube (e.g., FALCON™ tube). The cells areincubated at 28-30° C., 200-250 rpm for 3 h.

Aliquots of 250 μL are spread onto plates containing YM medium and 50μg/mL spectinomycin and incubated three days at 28-30° C. To increasethe number of transformants one of two optional steps can be performed:

Option 1: Overlay plates with 30 μL of 15 mg/mL rifampicin. LBA4404 hasa chromosomal resistance gene for rifampicin. This additional selectioneliminates some contaminating colonies observed when using poorerpreparations of LBA4404 competent cells.

Option 2: Perform two replicates of the electroporation to compensatefor poorer electrocompetent cells.

Identification of Transformants:

Four independent colonies are picked and streaked on plates containingAB minimal medium and 50 μg/mL spectinomycin for isolation of singlecolonies. The plates are incubated at 28° C. for two to three days. Asingle colony for each putative co-integrate is picked and inoculatedwith 4 mL of 10 g/L bactopeptone, 10 g/L yeast extract, 5 g/L sodiumchloride and 50 mg/L spectinomycin. The mixture is incubated for 24 h at28° C. with shaking. Plasmid DNA from 4 mL of culture is isolated usingQiagen® Miniprep and an optional Buffer PB wash. The DNA is eluted in 30μL. Aliquots of 2 μL are used to electroporate 20 μL of DH10b+20 μL oftwice distilled H₂O as per above. Optionally a 15 μL aliquot can be usedto transform 75-100 μL of INVITROGEN™ Library Efficiency DH5α. The cellsare spread on plates containing LB medium and 50 μg/mL spectinomycin andincubated at 37° C. overnight.

Three to four independent colonies are picked for each putativeco-integrate and inoculated 4 mL of 2×YT medium (10 g/L bactopeptone, 10g/L yeast extract, 5 g/L sodium chloride) with 50 μg/mL spectinomycin.The cells are incubated at 37° C. overnight with shaking. Next, isolatethe plasmid DNA from 4 mL of culture using QIAprep® Miniprep withoptional Buffer PB wash (elute in 50 4). Use 8 μL for digestion withSalI (using parental DNA and PHP10523 as controls). Three moredigestions using restriction enzymes BamHI, EcoRI, and HindIII areperformed for 4 plasmids that represent 2 putative co-integrates withcorrect SalI digestion pattern (using parental DNA and PHP10523 ascontrols). Electronic gels are recommended for comparison.

Example 13 Transformation of Maize Using Agrobacterium

Maize plants can be transformed to overexpress a validated Arabidopsislead gene or the corresponding homologs from various species in order toexamine the resulting phenotype.

Agrobacterium-mediated transformation of maize is performed essentiallyas described by Zhao et al. in Meth. Mol. Biol. 318:315-323 (2006) (seealso Zhao et al., Mol. Breed. 8:323-333 (2001) and U.S. Pat. No.5,981,840 issued Nov. 9, 1999, incorporated herein by reference). Thetransformation process involves bacterium innoculation, co-cultivation,resting, selection and plant regeneration.

1. Immature Embryo Preparation:

Immature maize embryos are dissected from caryopses and placed in a 2 mLmicrotube containing 2 mL PHI-A medium.

2. Agrobacterium Infection and Co-Cultivation of Immature Embryos:

2.1 Infection Step:

PHI-A medium of (1) is removed with 1 mL micropipettor, and 1 mL ofAgrobacterium suspension is added. The tube is gently inverted to mix.The mixture is incubated for 5 min at room temperature.

2.2 Co-Culture Step:

The Agrobacterium suspension is removed from the infection step with a 1mL micropipettor. Using a sterile spatula the embryos are scraped fromthe tube and transferred to a plate of PHI-B medium in a 100×15 mm Petridish. The embryos are oriented with the embryonic axis down on thesurface of the medium. Plates with the embryos are cultured at 20° C.,in darkness, for three days. L-Cysteine can be used in theco-cultivation phase. With the standard binary vector, theco-cultivation medium supplied with 100-400 mg/L L-cysteine is criticalfor recovering stable transgenic events.

3. Selection of Putative Transgenic Events:

To each plate of PHI-D medium in a 100×15 mm Petri dish, 10 embryos aretransferred, maintaining orientation and the dishes are sealed withparafilm. The plates are incubated in darkness at 28° C. Activelygrowing putative events, as pale yellow embryonic tissue, are expectedto be visible in six to eight weeks. Embryos that produce no events maybe brown and necrotic, and little friable tissue growth is evident.Putative transgenic embryonic tissue is subcultured to fresh PHI-Dplates at two-three week intervals, depending on growth rate. The eventsare recorded.

4. Regeneration of T0 Plants:

Embryonic tissue propagated on PHI-D medium is subcultured to PHI-Emedium (somatic embryo maturation medium), in 100×25 mm Petri dishes andincubated at 28° C., in darkness, until somatic embryos mature, forabout ten to eighteen days. Individual, matured somatic embryos withwell-defined scutellum and coleoptile are transferred to PHI-F embryogermination medium and incubated at 28° C. in the light (about 80 μEfrom cool white or equivalent fluorescent lamps). In seven to ten days,regenerated plants, about 10 cm tall, are potted in horticultural mixand hardened-off using standard horticultural methods.

Media for Plant Transformation:

-   -   1. PHI-A: 4 g/L CHU basal salts, 1.0 mL/L 1000× Eriksson's        vitamin mix, 0.5 mg/L thiamin HCl, 1.5 mg/L 2,4-D, 0.69 g/L        L-proline, 68.5 g/L sucrose, 36 g/L glucose, pH 5.2. Add 100 μM        acetosyringone (filter-sterilized).    -   2. PHI-B: PHI-A without glucose, increase 2,4-D to 2 mg/L,        reduce sucrose to 30 g/L and supplemente with 0.85 mg/L silver        nitrate (filter-sterilized), 3.0 g/L Gelrite®, 100 μM        acetosyringone (filter-sterilized), pH 5.8.    -   3. PHI-C: PHI-B without Gelrite® and acetosyringonee, reduce        2,4-D to 1.5 mg/L and supplemente with 8.0 g/L agar, 0.5 g/L        2-[N-morpholino]ethane-sulfonic acid (MES) buffer, 100 mg/L        carbenicillin (filter-sterilized).    -   4. PHI-D: PHI-C supplemented with 3 mg/L bialaphos        (filter-sterilized).    -   5. PHI-E: 4.3 g/L of Murashige and Skoog (MS) salts, (Gibco, BRL        11117-074), 0.5 mg/L nicotinic acid, 0.1 mg/L thiamine HCl, 0.5        mg/L pyridoxine HCl, 2.0 mg/L glycine, 0.1 g/L myo-inositol, 0.5        mg/L zeatin (Sigma, Cat. No. Z-0164), 1 mg/L indole acetic acid        (IAA), 26.4 μg/L abscisic acid (ABA), 60 g/L sucrose, 3 mg/L        bialaphos (filter-sterilized), 100 mg/L carbenicillin        (filter-sterilized), 8 g/L agar, pH 5.6.    -   6. PHI-F: PHI-E without zeatin, IAA, ABA; reduce sucrose to 40        g/L; replacing agar with 1.5 g/L Gelrite®; pH 5.6.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al., Bio/Technology 8:833-839 (1990)).

Transgenic T0 plants can be regenerated and their phenotype determined.T1 seed can be collected.

Furthermore, a recombinant DNA construct containing a validatedArabidopsis gene can be introduced into an elite maize inbred lineeither by direct transformation or introgression from a separatelytransformed line.

Transgenic plants, either inbred or hybrid, can undergo more vigorousfield-based experiments to study yield enhancement and/or stabilityunder water limiting and water non-limiting conditions.

Subsequent yield analysis can be done to determine whether plants thatcontain the validated Arabidopsis lead gene have an improvement in yieldperformance (under water limiting or non-limiting conditions), whencompared to the control (or reference) plants that do not contain thevalidated Arabidopsis lead gene. Specifically, water limiting conditionscan be imposed during the flowering and/or grain fill period for plantsthat contain the validated Arabidopsis lead gene and the control plants.Plants containing the validated Arabidopsis lead gene would have lessyield loss relative to the control plants, for example, at least 50%less yield loss, under water limiting conditions, or would haveincreased yield relative to the control plants under water non-limitingconditions.

Example 14A Preparation of Arabidopsis Lead Gene (At5g26030) ExpressionVector for Transformation of Maize

Using INVITROGEN's™ GATEWAY® technology, an LR Recombination Reactionwas performed with an entry clone (PHP31052) and a destination vector(PHP28647) to create the precursor plasmid PHP31079. The vector PHP31079contains the following expression cassettes:

1. Ubiquitin promoter::moPAT::PinII terminator; cassette expressing thePAT herbicide resistance gene used for selection during thetransformation process.

2. LTP2 promoter::DS-RED2::PinII terminator; cassette expressing theDS-RED color marker gene used for seed sorting.

3. Ubiquitin promoter::At5g26030::PinII terminator; cassetteoverexpressing the gene of interest, Arabidopsis ferrochelatase-I.

Example 14B Transformation of Maize with the Arabidopsis Lead Gene(At5g26030) Using Agrobacterium

The ferrochelatase-I expression cassette present in vector PHP31079 canbe introduced into a maize inbred line, or a transformable maize linederived from an elite maize inbred line, using Agrobacterium-mediatedtransformation as described in Examples 12 and 13.

Vector PHP31079 can be electroporated into the LBA4404 Agrobacteriumstrain containing vector PHP10523 (FIG. 7; SEQ ID NO:37) to create theco-integrate vector PHP31217. The co-integrate vector is formed byrecombination of the 2 plasmids, PHP31079 and PHP10523, through the COSrecombination sites contained on each vector. The co-integrate vectorPHP31217 contains the same 3 expression cassettes as above (Example 14A)in addition to other genes (TET, TET, TRFA, ORI terminator, CTL, ORI V,VIR C1, VIR C2, VIR G, VIR B) needed for the Agrobacterium strain andthe Agrobacterium-mediated transformation.

Example 15 Preparation of the Destination Vector PHP23236 forTransformation into Gaspe Flint Derived Maize Lines

Destination vector PHP23236 (FIG. 6, SEQ ID NO:36) was obtained bytransformation of Agrobacterium strain LBA4404 containing plasmidPHP10523 (FIG. 7, SEQ ID NO:37) with plasmid PHP23235 (FIG. 8, SEQ IDNO:38) and isolation of the resulting co-integration product.Destination vector PHP23236, can be used in a recombination reactionwith an entry clone as described in Example 16 to create a maizeexpression vector for transformation of Gaspe Flint-derived maize lines.

Example 16 Preparation of Plasmids for Transformation into Gaspe FlintDerived Maize Lines

Using the INVITROGEN™ GATEWAY® LR Recombination technology, the sameentry clone described in Example 5A, PHP31052, was directionally clonedinto the destination vector PHP23236 (SEQ ID NO:36; FIG. 6) to create anexpression vector, PHP31419. This expression vector contains the cDNA ofinterest, encoding AtFeC-I, under control of the UBI promoter and is aT-DNA binary vector for Agrobacterium-mediated transformation into cornas described, but not limited to, the examples described herein.

Using the INVITROGEN™ GATEWAY® LR Recombination technology, the sameentry clone described in Example 5B, PHP-Entry-At2g30390, wasdirectionally cloned into the destination vector PHP29634 to create anexpression vector, PHP33089. Destination vector PHP29634 is similar todestination vector PHP23236, however, destination vector PHP29634 hassite-specific recombination sites FRT1 and FRT87 and also encodes theGAT4602 selectable marker protein for selection of transformants usingglyphosate. This expression vector contains the cDNA of interest,encoding AtFeC-II, under control of the UBI promoter and is a T-DNAbinary vector for Agrobacterium-mediated transformation into corn asdescribed, but not limited to, the examples described herein.

Example 17 Transformation of Gaspe Flint Derived Maize Lines with aValidated Arabidopsis Lead Gene

Maize plants can be transformed to overexpress the Arabidopsis lead geneor the corresponding homologs from other species in order to examine theresulting phenotype.

Recipient Plants:

Recipient plant cells can be from a uniform maize line having a shortlife cycle (“fast cycling”), a reduced size, and high transformationpotential. Typical of these plant cells for maize are plant cells fromany of the publicly available Gaspe Flint (GBF) line varieties. Onepossible candidate plant line variety is the F1 hybrid of GBF×QTM (QuickTurnaround Maize, a publicly available form of Gaspe Flint selected forgrowth under greenhouse conditions) disclosed in Tomes et al. U.S.Patent Application Publication No. 2003/0221212. Transgenic plantsobtained from this line are of such a reduced size that they can begrown in four inch pots (¼ the space needed for a normal sized maizeplant) and mature in less than 2.5 months. (Traditionally 3.5 months isrequired to obtain transgenic T0 seed once the transgenic plants areacclimated to the greenhouse.) Another suitable line is a double haploidline of GS3 (a highly transformable line) X Gaspe Flint. Yet anothersuitable line is a transformable elite inbred line carrying a transgenewhich causes early flowering, reduced stature, or both.

Transformation Protocol:

Any suitable method may be used to introduce the transgenes into themaize cells, including but not limited to inoculation type proceduresusing Agrobacterium based vectors. Transformation may be performed onimmature embryos of the recipient (target) plant.

Precision Growth and Plant Tracking:

The event population of transgenic (T0) plants resulting from thetransformed maize embryos is grown in a controlled greenhouseenvironment using a modified randomized block design to reduce oreliminate environmental error. A randomized block design is a plantlayout in which the experimental plants are divided into groups (e.g.,thirty plants per group), referred to as blocks, and each plant israndomly assigned a location with the block.

For a group of thirty plants, twenty-four transformed, experimentalplants and six control plants (plants with a set phenotype)(collectively, a “replicate group”) are placed in pots which arearranged in an array (a.k.a. a replicate group or block) on a tablelocated inside a greenhouse. Each plant, control or experimental, israndomly assigned to a location with the block which is mapped to aunique, physical greenhouse location as well as to the replicate group.Multiple replicate groups of thirty plants each may be grown in the samegreenhouse in a single experiment. The layout (arrangement) of thereplicate groups should be determined to minimize space requirements aswell as environmental effects within the greenhouse. Such a layout maybe referred to as a compressed greenhouse layout.

An alternative to the addition of a specific control group is toidentify those transgenic plants that do not express the gene ofinterest. A variety of techniques such as RT-PCR can be applied toquantitatively assess the expression level of the introduced gene. T0plants that do not express the transgene can be compared to those whichdo.

Each plant in the event population is identified and tracked throughoutthe evaluation process, and the data gathered from that plant isautomatically associated with that plant so that the gathered data canbe associated with the transgene carried by the plant. For example, eachplant container can have a machine readable label (such as a UniversalProduct Code (UPC) bar code) which includes information about the plantidentity, which in turn is correlated to a greenhouse location so thatdata obtained from the plant can be automatically associated with thatplant.

Alternatively any efficient, machine readable, plant identificationsystem can be used, such as two-dimensional matrix codes or even radiofrequency identification tags (RFID) in which the data is received andinterpreted by a radio frequency receiver/processor. See U.S. PublishedPatent Application No. 2004/0122592, incorporated herein by reference.

Phenotypic Analysis Using Three-Dimensional Imaging:

Each greenhouse plant in the T0 event population, including any controlplants, is analyzed for agronomic characteristics of interest, and theagronomic data for each plant is recorded or stored in a manner so thatit is associated with the identifying data (see above) for that plant.Confirmation of a phenotype (gene effect) can be accomplished in the T1generation with a similar experimental design to that described above.

The T0 plants are analyzed at the phenotypic level using quantitative,non-destructive imaging technology throughout the plant's entiregreenhouse life cycle to assess the traits of interest. A digitalimaging analyzer may be used for automatic multi-dimensional analyzingof total plants. The imaging may be done inside the greenhouse. Twocamera systems, located at the top and side, and an apparatus to rotatethe plant, are used to view and image plants from all sides. Images areacquired from the top, front and side of each plant. All three imagestogether provide sufficient information to evaluate the biomass, sizeand morphology of each plant.

Due to the change in size of the plants from the time the first leafappears from the soil to the time the plants are at the end of theirdevelopment, the early stages of plant development are best documentedwith a higher magnification from the top. This may be accomplished byusing a motorized zoom lens system that is fully controlled by theimaging software.

In a single imaging analysis operation, the following events occur: (1)the plant is conveyed inside the analyzer area, rotated 360 degrees soits machine readable label can be read, and left at rest until itsleaves stop moving; (2) the side image is taken and entered into adatabase; (3) the plant is rotated 90 degrees and again left at restuntil its leaves stop moving, and (4) the plant is transported out ofthe analyzer.

Plants are allowed at least six hours of darkness per twenty four hourperiod in order to have a normal day/night cycle.

Imaging Instrumentation:

Any suitable imaging instrumentation may be used, including but notlimited to light spectrum digital imaging instrumentation commerciallyavailable from LemnaTec GmbH of Wurselen, Germany. The images are takenand analyzed with a LemnaTec Scanalyzer HTS LT-0001-2 having a ½″ ITProgressive Scan IEE CCD imaging device. The imaging cameras may beequipped with a motor zoom, motor aperture and motor focus. All camerasettings may be made using LemnaTec software. For example, theinstrumental variance of the imaging analyzer is less than about 5% formajor components and less than about 10% for minor components.

Software:

The imaging analysis system comprises a LemnaTec HTS Bonit softwareprogram for color and architecture analysis and a server database forstoring data from about 500,000 analyses, including the analysis dates.The original images and the analyzed images are stored together to allowthe user to do as much reanalyzing as desired. The database can beconnected to the imaging hardware for automatic data collection andstorage. A variety of commercially available software systems (e.g.Matlab, others) can be used for quantitative interpretation of theimaging data, and any of these software systems can be applied to theimage data set.

Conveyor System:

A conveyor system with a plant rotating device may be used to transportthe plants to the imaging area and rotate them during imaging. Forexample, up to four plants, each with a maximum height of 1.5 m, areloaded onto cars that travel over the circulating conveyor system andthrough the imaging measurement area. In this case the total footprintof the unit (imaging analyzer and conveyor loop) is about 5 m×5 m.

The conveyor system can be enlarged to accommodate more plants at atime. The plants are transported along the conveyor loop to the imagingarea and are analyzed for up to 50 seconds per plant. Three views of theplant are taken. The conveyor system, as well as the imaging equipment,should be capable of being used in greenhouse environmental conditions.

Illumination:

Any suitable mode of illumination may be used for the image acquisition.For example, a top light above a black background can be used.Alternatively, a combination of top- and backlight using a whitebackground can be used. The illuminated area should be housed to ensureconstant illumination conditions. The housing should be longer than themeasurement area so that constant light conditions prevail withoutrequiring the opening and closing or doors. Alternatively, theillumination can be varied to cause excitation of either transgene(e.g., green fluorescent protein (GFP), red fluorescent protein (RFP))or endogenous (e.g. Chlorophyll) fluorophores.

Biomass Estimation Based on Three-Dimensional Imaging:

For best estimation of biomass the plant images should be taken from atleast three axes, for example, the top and two side (sides 1 and 2)views. These images are then analyzed to separate the plant from thebackground, pot and pollen control bag (if applicable). The volume ofthe plant can be estimated by the calculation:Volume(voxels)=√{square root over (TopArea(pixels))}×√{square root over(Side1Area(pixels))}×√{square root over (Side2Area(pixels))}

In the equation above the units of volume and area are “arbitraryunits”. Arbitrary units are entirely sufficient to detect gene effectson plant size and growth in this system because what is desired is todetect differences (both positive-larger and negative-smaller) from theexperimental mean, or control mean. The arbitrary units of size (e.g.area) may be trivially converted to physical measurements by theaddition of a physical reference to the imaging process. For instance, aphysical reference of known area can be included in both top and sideimaging processes. Based on the area of these physical references aconversion factor can be determined to allow conversion from pixels to aunit of area such as square centimeters (cm²). The physical referencemay or may not be an independent sample. For instance, the pot, with aknown diameter and height, could serve as an adequate physicalreference.

Color Classification:

The imaging technology may also be used to determine plant color and toassign plant colors to various color classes. The assignment of imagecolors to color classes is an inherent feature of the LemnaTec software.With other image analysis software systems color classification may bedetermined by a variety of computational approaches.

For the determination of plant size and growth parameters, a usefulclassification scheme is to define a simple color scheme including twoor three shades of green and, in addition, a color class for chlorosis,necrosis and bleaching, should these conditions occur. A backgroundcolor class which includes non plant colors in the image (for examplepot and soil colors) is also used and these pixels are specificallyexcluded from the determination of size. The plants are analyzed undercontrolled constant illumination so that any change within one plantover time, or between plants or different batches of plants (e.g.seasonal differences) can be quantified.

In addition to its usefulness in determining plant size growth, colorclassification can be used to assess other yield component traits. Forthese other yield component traits additional color classificationschemes may be used. For instance, the trait known as “staygreen”, whichhas been associated with improvements in yield, may be assessed by acolor classification that separates shades of green from shades ofyellow and brown (which are indicative of senescing tissues). Byapplying this color classification to images taken toward the end of theT0 or T1 plants' life cycle, plants that have increased amounts of greencolors relative to yellow and brown colors (expressed, for instance, asGreen/Yellow Ratio) may be identified. Plants with a significantdifference in this Green/Yellow ratio can be identified as carryingtransgenes which impact this important agronomic trait.

The skilled plant biologist will recognize that other plant colors arisewhich can indicate plant health or stress response (for instanceanthocyanins), and that other color classification schemes can providefurther measures of gene action in traits related to these responses.

Plant Architecture Analysis:

Transgenes which modify plant architecture parameters may also beidentified using the present invention, including such parameters asmaximum height and width, internodal distances, angle between leaves andstem, number of leaves starting at nodes and leaf length. The LemnaTecsystem software may be used to determine plant architecture as follows.The plant is reduced to its main geometric architecture in a firstimaging step and then, based on this image, parameterized identificationof the different architecture parameters can be performed. Transgenesthat modify any of these architecture parameters either singly or incombination can be identified by applying the statistical approachespreviously described.

Pollen Shed Date:

Pollen shed date is an important parameter to be analyzed in atransformed plant, and may be determined by the first appearance on theplant of an active male flower. To find the male flower object, theupper end of the stem is classified by color to detect yellow or violetanthers. This color classification analysis is then used to define anactive flower, which in turn can be used to calculate pollen shed date.

Alternatively, pollen shed date and other easily visually detected plantattributes (e.g. pollination date, first silk date) can be recorded bythe personnel responsible for performing plant care. To maximize dataintegrity and process efficiency this data is tracked by utilizing thesame barcodes utilized by the LemnaTec light spectrum digital analyzingdevice. A computer with a barcode reader, a palm device, or a notebookPC may be used for ease of data capture recording time of observation,plant identifier, and the operator who captured the data.

Orientation of the Plants:

Mature maize plants grown at densities approximating commercial plantingoften have a planar architecture. That is, the plant has a clearlydiscernable broad side, and a narrow side. The image of the plant fromthe broadside is determined. To each plant a well defined basicorientation is assigned to obtain the maximum difference between thebroadside and edgewise images. The top image is used to determine themain axis of the plant, and an additional rotating device is used toturn the plant to the appropriate orientation prior to starting the mainimage acquisition.

Example 18 Screening of Gaspe Flint Derived Maize Lines for DroughtTolerance

Transgenic Gaspe Flint derived maize lines containing the candidate genecan be screened for tolerance to drought stress in the following manner.

Transgenic maize plants are subjected to well-watered conditions(control) and to drought-stressed conditions. Transgenic maize plantsare screened at the T1 stage or later.

Stress is imposed starting at 10 to 14 days after sowing (DAS) or 7 daysafter transplanting, and is continued through to silking. Pots arewatered by an automated system fitted to timers to provide watering at25 or 50% of field capacity during the entire period of drought-stresstreatment. The intensity and duration of this stress will allowidentification of the impact on vegetative growth as well as on theanthesis-silking interval.

Potting Mixture:

A mixture of ⅓ TURFACE® (Profile Products LLC, IL, USA), ⅓ sand and ⅓SB300 (Sun Gro Horticulture, WA, USA) can be used. The SB300 can bereplaced with Fafard Fine-Germ (Conrad Fafard, Inc., MA, USA) and theproportion of sand in the mixture can be reduced. Thus, a final pottingmixture can be ⅜ (37.5%) TURFACE®, ⅜ (37.5%) Fafard and ¼ (25%) sand.

Field Capacity Determination:

The weight of the soil mixture (w1) to be used in one S200 pot (minusthe pot weight) is measured. If all components of the soil mix are notdry, the soil is dried at 100° C. to constant weight before determiningw1. The soil in the pot is watered to full saturation and all thegravitational water is allowed to drain out. The weight of the soil (w2)after all gravitational water has seeped out (minus the pot weight) isdetermined. Field capacity is the weight of the water remaining in thesoil obtained as w2-w1. It can be written as a percentage of theoven-dry soil weight.

Stress Treatment:

During the early part of plant growth (10 DAS to 21 DAS), thewell-watered control has a daily watering of 75% field capacity and thedrought-stress treatment has a daily watering of 25% field capacity,both as a single daily dose at or around 10 AM. As the plants growbigger, by 21 DAS, it will become necessary to increase the dailywatering of the well-watered control to full field capacity and thedrought stress treatment to 50% field capacity.

Nutrient Solution:

A modified Hoagland's solution at 1/16 dilution with tap water is usedfor irrigation.

TABLE 8 Preparation of 20 L of Modified Hoagland's Solution Using theFollowing Recipe: Component Amount/20 L 10X Micronutrient Solution 16 mLKH₂PO₄ (MW: 136.02) 22 g MgSO₄ (MW: 120.36) 77 g KNO₃ (MW: 101.2) 129.5g Ca(NO₃)₂•4H₂0 (MW: 236.15) 151 g NH₄NO₃ (MW: 80.04) 25.6 g Sprint 330(Iron chelate) 32 g

TABLE 9 Preparation of 1 L of 10X Micronutrient Solution Using theFollowing Recipe: Component mg/L Concentration H₃BO₃ 1854 30 mMMnCl₂•4H₂0 1980 10 mM ZnSO₄•7H₂0 2874 10 mM CuSO₄•5H₂0 250  1 mMH₂MoO₄•H₂0 242  1 mM

Fertilizer grade KNO₃ is used.

It is useful to add half a teaspoon of OSMOCOTE® (NPK 15:9:12) to thepot at the time of transplanting or after emergence (The ScottsMiracle-Gro Company, OH, USA).

Border Plants:

Place a row of border plants on bench-edges adjacent to the glass wallsof the greenhouse or adjacent to other potential causes ofmicroenvironment variability such as a cooler fan.

Automation:

Watering can be done using PVC pipes with drilled holes to supply waterto systematically positioned pots using a siphoning device. Irrigationscheduling can be done using timers.

Statistical Analysis:

Mean values for plant size, color and chlorophyll fluorescence recordedon transgenic events under different stress treatments will be exportedto Spotfire (Spotfire, Inc., MA, USA). Treatment means will be evaluatedfor differences using Analysis of Variance.

Replications:

Eight to ten individual plants are used per treatment per event.

Observations Made:

Lemnatec measurements are made three times a week throughout growth tocapture plant-growth rate. Leaf color determinations are made threetimes a week throughout the stress period using Lemnatec. Chlorophyllfluorescence is recorded as PhiPSII (which is indicative of theoperating quantum efficiency of photosystem II photochemistry) andFv′/Fm′ (which is the maximum efficiency of photosystem II) two to fourtimes during the experimental period, starting at 11 AM on themeasurement days, using the Hansatech FMS2 instrument (LemnaTec GmbH,Wurselen, Germany). Measurements are started during the stress period atthe beginning of visible drought stress symptoms, namely, leaf greyingand the start of leaf rolling until the end of the experiment andmeasurements are recorded on the youngest most fully expanded leaf. Thedates of tasseling and silking on individual plants are recorded, andthe ASI is computed.

The above methods may be used to select transgenic plants with increaseddrought tolerance when compared to a control plant not comprising saidrecombinant DNA construct.

Example 19 Yield Analysis of Maize Lines with the Arabidopsis Lead Gene

A recombinant DNA construct containing a validated Arabidopsis gene canbe introduced into an elite maize inbred line either by directtransformation or introgression from a separately transformed line.

Transgenic plants, either inbred or hybrid, can undergo more vigorousfield-based experiments to study yield enhancement and/or stabilityunder well-watered and water-limiting conditions.

Subsequent yield analysis can be done to determine whether plants thatcontain the validated Arabidopsis lead gene have an improvement in yieldperformance under water-limiting conditions, when compared to thecontrol plants that do not contain the validated Arabidopsis lead gene.Specifically, drought conditions can be imposed during the floweringand/or grain fill period for plants that contain the validatedArabidopsis lead gene and the control plants. Reduction in yield can bemeasured for both. Plants containing the validated Arabidopsis lead genehave less yield loss relative to the control plants, for example, atleast 50% less yield loss.

The above method may be used to select transgenic plants with increasedyield, under water-limiting conditions and/or well-watered conditions,when compared to a control plant not comprising said recombinant DNAconstruct.

Example 20A Preparation of Maize Ferrochelatase-I Lead Gene ExpressionVector for Transformation of Maize

Clone cfp5n.pk009.j16 encodes a maize ferrochelatase-I proteindesignated “ZmFeC-Ia” (SEQ ID NO:10). The protein-coding region of clonecfp5n.pk009.j16 was introduced into the INVITROGEN™ vector pENTR/D-TOPO®to create entry clone PHP30949 (SEQ ID NO:55).

Using INVITROGEN's™ GATEWAY® technology, an LR Recombination Reactionwas performed with an entry clone (PHP30949) and a destination vector(PHP28647) to create the precursor plasmid PHP30963. The vector PHP30963contains the following expression cassettes:

1. Ubiquitin promoter::moPAT::PinII terminator; cassette expressing thePAT herbicide resistance gene used for selection during thetransformation process.

2. LTP2 promoter::DS-RED2::PinII terminator; cassette expressing theDS-RED color marker gene used for seed sorting.

3. Ubiquitin promoter::ZmFeC-Ia::PinII terminator; cassetteoverexpressing the gene of interest, maize ferrochelatase-I.

Example 20B Transformation of Maize with Maize Ferrochelatase-I LeadGene Using Agrobacterium

The ZmFeC-Ia expression cassette present in vector PHP30963 can beintroduced into a maize inbred line, or a transformable maize linederived from an elite maize inbred line, using Agrobacterium-mediatedtransformation as described in Examples 12 and 13.

Vector PHP30963 can be electroporated into the LBA4404 Agrobacteriumstrain containing vector PHP10523 (FIG. 7; SEQ ID NO:37) to create theco-integrate vector PHP30976. The co-integrate vector is formed byrecombination of the 2 plasmids, PHP30963 and PHP10523, through the COSrecombination sites contained on each vector. The co-integrate vectorPHP30976 contains the same 3 expression cassettes as above (Example 14A)in addition to other genes (TET, TET, TRFA, ORI terminator, CTL, ORI V,VIR C1, VIR C2, VIR G, VIR B) needed for the Agrobacterium strain andthe Agrobacterium-mediated transformation.

Example 21 Preparation of Maize Expression Plasmids for Transformationinto Gaspe Flint Derived Maize Lines

Clone cfp5n.pk009.j16 encodes a complete maize ferrochelatase-I homologdesignated “ZmFeC-Ia” (SEQ ID NO:10). Clone cfp3n.pk004.f12 encodes adifferent complete maize ferrochelatase-I homolog designated “ZmFeC-Ib”(SEQ ID NO:8). Clone cfp5n.pk064.n7 also encodes the ZmFeC-Ib protein(SEQ ID NO:8), however the nucleotide sequence of cfp5n.pk064.n7 (SEQ IDNO:56) differs from the nucleotide sequence of cfp3n.pk004.f12 (SEQ IDNO:7) in that it contains a 12 base pair insertion in the 5′-UTR, 8nucleotides before the ATG start codon.

Using the INVITROGEN™ GATEWAY® Recombination technology described inExample 9, the three clones encoding maize ferrochelatase-I homologswere directionally cloned into the destination vector PHP23236 (SEQ IDNO:36; FIG. 6) to create the expression vectors listed in Table 10. Eachexpression vector contains the cDNA of interest under control of the UBIpromoter and is a T-DNA binary vector for Agrobacterium-mediatedtransformation into corn as described, but not limited to, the examplesdescribed herein.

TABLE 10 Maize Ferrochelatase-I Expression Vectors SEQ ID NO: ExpressionProtein Clone Origin (Amino Acid) Vector ZmFeC-Ia cfp5n.pk009.j16 (FIS)10 PHP30829 ZmFeC-Ib cfp3n.pk004.f12 (FIS) 8 PHP30745 ZmFeC-Ibcfp5n.pk064.n7 (FIS) 8 PHP30761

Example 22 Transformation and Evaluation of Gaspe Flint Derived MaizeLines for Drought Tolerance

Gaspe Flint derived maize lines were transformed via Agrobacterium usingthe following plasmid DNAs: PHP31419 (AtFeC-I; At5g26030); PHP33089(AtFeC-II; At2g30390); PHP30829 (ZmFeC-Ia; cfp5n.pk009.j16); PHP30745(ZmFeC-Ib; cfp3n.pk004.f12) and PHP30761 (ZmFeC-Ib; cfp5n.pk064.n7).Four transformation events for each plasmid construct were evaluated fordrought tolerance in the following manner. For plant growth, the soilmixture consisted of ⅓ TURFACE®, ⅓ SB300 and ⅓ sand. All pots werefilled with the same amount of soil±10 grams. Pots were brought up to100% field capacity (“FC”) by hand watering. All plants were maintainedat 60% FC using a 20-10-20 (N-P-K) 125 ppm N nutrient solution.Throughout the experiment pH was monitored at least three times weeklyfor each table. Starting at 13 days after planting (DAP), the experimentwas divided into two treatment groups, well watered and reduce watered.All plants comprising the reduced watered treatment were maintained at40% FC while plants in the well watered treatment were maintained at 80%FC. Reduced watered plants were grown for 10 days under chronic droughtstress conditions (40% FC). All plants were imaged daily throughoutchronic stress period. Plants were sampled for metabolic profilinganalyses at the end of chronic drought period, 22 DAP. At the conclusionof the chronic stress period all plants were imaged and measured forchlorophyll fluorescence. Reduced watered plants were subjected to asevere drought stress period followed by a recovery period, 23-31 DAPand 32-34 DAP respectively. During the severe drought stress, water andnutrients were withheld until the plants reached 8% FC. At theconclusion of severe stress and recovery periods all plants were againimaged and measured for chlorophyll fluorescence. The probability of agreater Student's t Test was calculated for each transgenic meancompared to the appropriate null mean (either segregant null orconstruct null). A minimum (P<t) of 0.1 was used as a cut off for astatistically significant result.

Tables 11-20 show the variables for each transgenic event that weresignificantly altered, as compared to the segregant nulls. A “positiveeffect” was defined as statistically significant improvement in thatvariable for the transgenic event relative to the null control. A“negative effect” was defined as a statistically significant improvementin that variable for the null control relative to the transgenic event.Tables 11, 13, 15, 17, and 19 present the number of variable with asignificant change for individual events transformed with each of thefive plasmid DNA constructs. Tables 12, 14, 16, 18 and 20 present thenumber of events for each construct that showed a significant change foreach individual variable.

TABLE 11 Number of Variables with a Significant Change* for IndividualEvents Transformed with PHP31419 Encoding AtFeC-I (At5g26030) ReducedWater Well Watered Positive Negative Positive Negative Event EffectEffect Effect Effect EA2392.498.1.2 2 1 4 0 EA2392.498.1.3 3 1 1 3EA2392.498.1.5 0 0 3 0 EA2392.498.1.6 12 0 3 0 *P-value less than orequal to 0.1

TABLE 12 Number of Events Transformed with PHP31419 Encoding AtFeC-I(At5g26030) with a Significant Change* for Individual Variables ReducedWater Well Watered Positive Negative Positive Negative Variable EffectEffect Effect Effect % area chg_start 1 0 1 0 chronic - acute2 % areachg_start 1 0 2 0 chronic - end chronic % area chg_start 1 0 1 0chronic - harvest % area chg_start chronic - 1 0 1 1 recovery24hrfv/fm_acute1 2 0 2 0 fv/fm_acute2 1 0 1 0 leaf rolling_harvest 2 0 0 0leaf 2 0 1 0 rolling_recovery24hr psii_acute1 2 0 2 0 psii_acute2 1 0 00 sgr - r2 > 0.9 2 0 0 0 shoot dry weight 0 1 0 1 shoot fresh weight 1 10 1 *P-value less than or equal to 0.1

TABLE 13 Number of Variables with a Significant Change* for IndividualEvents Transformed with PHP33089 Encoding AtFeC-II (At2g30390) ReducedWater Well Watered Positive Negative Positive Negative Event EffectEffect Effect Effect EA2534.088.2.2 0 3 1 0 EA2534.088.2.4 3 1 2 2EA2534.088.2.5 6 0 2 2 EA2534.088.2.8 1 3 0 2 *P-value less than orequal to 0.1

TABLE 14 Number of Events Transformed with PHP33089 Encoding AtFeC-II(At2g30390) with a Significant Change* for Individual Variables ReducedWater Well Watered Positive Negative Positive Negative Variable EffectEffect Effect Effect % area chg_start 2 0 1 0 chronic - end chronic %area chg_start 1 2 0 0 chronic - end severe % area chg_start 0 1 0 0chronic - recovery48hr fv/fm_end severe 1 2 1 0 fv/fm_recovery48hr 1 0 01 fv/fm_start severe 1 0 0 0 leaf 0 0 1 1 rolling_recovery24hr psii_endsevere 1 2 1 0 psii_recovery48hr 1 0 0 2 psii_start severe 1 0 1 2 sgr -r2 > 0.9 0 0 0 0 shoot dry weight 1 0 0 0 shoot fresh weight 0 0 0 0*P-value less than or equal to 0.1

TABLE 15 Number of Variables with a Significant Change* for IndividualEvents Transformed with PHP30829 Encoding MzFeC-Ia (cfp5n.pk009.j16)Reduced Water Well Watered Positive Negative Positive Negative EventEffect Effect Effect Effect EA2391.472.1.10 0 5 3 3 EA2391.472.1.2 0 2 61 EA2391.472.1.3 0 4 2 0 EA2391.472.1.4 2 2 3 0 *P-value less than orequal to 0.1

TABLE 16 Number of Events Transformed with PHP30829 Encoding MzFeC-Ia(cfp5n.pk009.j16) with a Significant Change* for Individual VariablesReduced Water Well Watered Positive Negative Positive Negative VariableEffect Effect Effect Effect % area chg_start chronic - 0 2 0 0 endchronic % area chg_start chronic - 0 1 3 0 harvest % area chg_startchronic - 0 0 0 0 recovery24hr % area chg_start chronic - 0 1 0 0recovery48hr fv/fm_acute1 0 0 2 1 fv/fm_acute2 1 1 2 0 leafrolling_recovery24hr 0 1 1 0 leaf rolling_recovery48hr 0 2 1 0psii_acute1 0 0 2 1 psii_acute2 1 1 1 1 sgr - r2 > 0.9 0 2 1 0 shoot dryweight 0 1 0 1 shoot fresh weight 0 1 1 0 *P-value less than or equal to0.1

TABLE 17 Number of Variables with a Significant Change* for IndividualEvents Transformed with PHP30745 Encoding MzFeC-Ib (cfp3n.pk004.f12)Reduced Water Well Watered Positive Negative Positive Negative EventEffect Effect Effect Effect EA2392.447.1.1 4 1 4 0 EA2392.447.1.3 0 3 27 EA2392.447.1.5 0 0 2 1 EA2392.447.1.9 2 4 4 1 *P-value less than orequal to 0.1

TABLE 18 Number of Events Transformed with PHP30745 Encoding MzFeC-Ib(cfp3n.pk004.f12) with a Significant Change* for Individual VariablesReduced Water Well Watered Positive Negative Positive Negative VariableEffect Effect Effect Effect % area chg_start 1 0 3 1 chronic - endchronic % area chg_start 0 0 2 1 chronic - end severe % area chg_start 00 2 1 chronic - recovery48hr fv/fm_end severe 2 0 0 0 fv/fm_recovery48hr1 1 0 1 fv/fm_start severe 0 1 0 1 leaf rolling_recovery48hr 0 0 0 0psii_end severe 0 0 0 0 psii_recovery48hr 0 1 1 2 psii_start severe 0 20 1 shoot dry weight 2 0 2 1 shoot fresh weight 2 1 2 0 *P-value lessthan or equal to 0.1

TABLE 19 Number of Variables with a Significant Change* for IndividualEvents Transformed with PHP30761 Encoding MzFeC-Ib (cfp5n.pk064.n7)Reduced Water Well Watered Positive Negative Positive Negative EventEffect Effect Effect Effect EA2392.441.1.1 0 0 0 1 EA2392.441.1.2 0 0 20 EA2392.441.1.4 5 0 7 3 EA2392.441.1.7 5 3 0 0 *P-value less than orequal to 0.1

TABLE 20 Number of Events Transformed with PHP30761 Encoding MzFeC-Ib(cfp5n.pk064.n7) with a Significant Change* for Individual VariablesReduced Water Well Watered Positive Negative Positive Negative VariableEffect Effect Effect Effect % area chg_start 0 1 0 1 chronic - endchronic % area chg_start 0 1 0 0 chronic - end severe % area chg_start 00 0 0 chronic - recovery48hr fv/fm_end severe 2 0 1 0 fv/fm_recovery48hr2 0 1 0 fv/fm_start severe 0 0 2 0 leaf rolling_recovery48hr 1 0 1 0psii_end severe 2 0 1 0 psii_recovery48hr 2 0 1 0 psii_start severe 1 01 0 sgr - r2 > 0.9 0 0 0 1 shoot dry weight 0 1 1 2 shoot fresh weight 00 0 0 *P-value less than or equal to 0.1

For each of the five constructs evaluated, the statistical valueassociated with each improved variable is presented in FIGS. 14A-23. Asignificant positive result had a P-value of less than or equal to 0.1.The results for individual transformed maize lines are presented inFIGS. 14A-14B, 16A-16B, 18A-18B, 20A-20B and 22A-22B. The summaryevaluations for each of the five constructs are presented in FIGS. 15,17, 19, 21, and 23.

Example 23 Screening of Inbred Derived Maize Lines for Drought Tolerance

A transformable maize line derived from an elite maize inbred line wastransformed with PHP30829 which encodes the maize ferrochelatase-Iprotein, MzFeC-1a (SEQ ID NO:10). Seed of transgenic events from thePHP30829 transformation were separated into transgenic and null seedusing a seed color marker. The Fv′/Fm′ and Phi PSII data were collectedfrom a drought seedling assay following a procedure similar to the onedescribed in Example 18 with the following modifications: an elite maizehybrid seedling was used for the assay instead of a Gaspe Flint derivedmaize line; and the chlorophyll fluorescence data were collected onlyduring recovery from moderate drought stress (or chronic droughtstress). The experiment was designed and analyzed as a RandomizedComplete Block Design with the events including the construct null astreatments. A number of individual events were found to have higherFv′/Fm′ and/or Phi PSII values during drought stress relative to thenull segregant control. The data is presented in the Table below.

TABLE 21 Fv′/Fm′ and Phi PSII Values For Individual Events TransformedWith PHP30829 Encoding MzFeC-Ia (cfp5n.pk009.j16) Event Fv′/Fm′ Phi PSIIE7733.78.1.1 0.421 0.330^(a) E7733.78.1.2 0.462 0.407^(a) E7733.78.2.200.520^(a) 0.420^(a) E7733.78.4.1 0.428 0.339^(a) E7733.78.2.10 0.475^(a)0.398^(a) E7733.78.2.2 0.360^(b) 0.284 E7733.78.2.6 0.373^(b) 0.289E7733.78.2.7 0.469^(a) 0.392^(a) 30976-CN^(c) 0.392 0.314 ^(a)Positiveand statistically significant at the 0.1 level of confidence^(b)Negative and statistically significant at the 0.1 level ofconfidence ^(c)Null segregant used as a control

Example 24 Transformation and Evaluation of Soybean with SoybeanHomologs of Validated Lead Genes

Based on homology searches, one or several candidate soybean homologs ofvalidated Arabidopsis lead genes can be identified and also be assessedfor their ability to enhance drought tolerance in soybean. Vectorconstruction, plant transformation and phenotypic analysis will besimilar to that in previously described Examples.

Example 25 Transformation of Arabidopsis with Maize and Soybean Homologsof Validated Lead Genes

Soybean and maize homologs to validated Arabidopsis lead genes can betransformed into Arabidopsis under control of the 35S promoter andassessed for their ability to enhance drought tolerance in Arabidopsis.Vector construction, plant transformation and phenotypic analysis willbe similar to that in previously described Examples.

What is claimed is:
 1. A recombinant DNA construct comprising apolynucleotide operably linked to at least one heterologous regulatorysequence, wherein the polynucleotide comprises: (a) a nucleotidesequence encoding a polypeptide with ferrochelatase activity, whereinthe polypeptide has an amino acid sequence of at least 92% sequenceidentity, based on the Clustal V method of alignment with pairwisealignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5, when compared to SEQ ID NO:24; or (b) the fullcomplement of the nucleotide sequence of (a).
 2. The recombinant DNAconstruct of claim 1, wherein the polypeptide has an amino acid sequenceof at least 95% sequence identity, based on the Clustal V method ofalignment with the pairwise alignment default parameters, when comparedto SEQ ID NO:24.
 3. The recombinant DNA construct of claim 1, whereinthe amino acid sequence of the polypeptide comprises SEQ ID NO:24. 4.The recombinant DNA construct of claim 1 wherein the nucleotide sequencecomprises SEQ ID NO:23.
 5. A vector comprising the recombinant DNAconstruct of claim
 1. 6. A cell comprising the recombinant DNA constructof claim 1, wherein the cell is selected from the group consisting of: abacterial cell, a yeast cell, an insect cell and a plant cell.
 7. Aplant or seed comprising the recombinant DNA construct of claim
 1. 8.The plant or seed of claim 7, wherein the plant or seed is selected fromthe group consisting of: maize, soybean, sunflower, sorghum, canola,wheat, alfalfa, cotton, rice, barley and millet.